Value-assigned solutions of lipoprotein-associated phospholipase a2 having a long shelf-life

ABSTRACT

Value-assigned solutions having predetermined concentrations of recombinant Lp-PLA2 are described herein. In particular, described herein are solutions of rLp-PLA2 that are stable for an extended period of time. Kits and assays include these calibration solutions, as well as methods of making and using them are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 14/279,106, filed May 15, 2014, and titled “VALUE-ASSIGNEDSOLUTIONS OF LIPOPROTEIN-ASSOCIATED PHOSPHOLIPASE A2 HAVING A LONGSHELF-LIFE”, Publication No. US-2015-0086998-A1, which claims priorityU.S. Provisional Patent Application No. 61/881,881, filed on Sep. 24,2013, and titled “CALIBRATION STANDARDS FOR THE DETECTION OFLIPOPROTEIN-ASSOCIATED PHOSPHOLIPASE A2”. This application is hereinincorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 13, 2014, isnamed 12248-702.200_SL.txt and is 12,023 bytes in size.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

FIELD

Described herein are compositions, kits, assays and methods of makingand using them including calibration solutions having a long shelf-lifethat are stably maintain a predetermined level of functional,properly-folded lipoprotein-associated phospholipase A2 (Lp-PLA₂) overan extended period of time, and specifically the use of such calibrationsolutions as calibration standards in assays, e.g. ELISA (mass) assays,activity assays, or the like, for detection of Lp-PLA₂.

BACKGROUND

Lipoprotein-associated phospholipase A2 (Lp-PLA2 or LP-PLA2) is anenzymatically active 50 kD protein that has been associated withCoronary vascular disease (CVD) including coronary heart disease (CHD)and stroke. Lp-PLA2 has been previously identified and characterized inthe literature by Tew et al. (1996) Arterioscler. Thromb. Vasc. Biol.16:591-599, Tjoelker, et al. (1995) Nature 374(6522):549-53), andCaslake et al. (2000) Atherosclerosis 150(2): 413-9. In addition, theprotein, assays and methods of use have been described in the patentliterature WO 95/00649-A1: U.S. Pat. Nos. 5,981,252, 5,968,818,6,177,257, 7,052,862, 7,045,329, 7,217,535, 7,416,853; WO 00/24910-A1:U.S. Pat. No. 5,532,152; 5,605,801; 5,641,669; 5,656,431; 5,698,403;5,977,308; and 5,847,088; WO 04/089184; WO 05/001416: U.S. Pat. No.7,531,316; WO 05/074604; WO 05/113797; the contents of which are herebyincorporated by reference in their entirety. Lp-PLA2 is expressed bymacrophages, with increased expression in atherosclerotic lesions(Hakkinen (1999) Arterioscler Thromb Vase Biol 19(12): 2909-17). Lp-PLA2circulates in the blood bound mainly to LDL, co-purifies with LDL, andis responsible for >95% of the phospholipase activity associated withLDL (Caslake 2000).

There are a handful of tests, both “mass” (e.g., ELISA-type) assays andactivity (e.g., enzymatic activity) assays that have been described. Forexample, the United States Food and Drug Administration (FDA) hasgranted clearance for the PLAC® Test (diaDexus, South San Francisco,Calif.) for the quantitative determination of Lp-PLA2 in human plasma orserum, to be used in conjunction with clinical evaluation and patientrisk assessment as an aid in predicting risk for coronary heart disease,and ischemic stroke associated with atherosclerosis. Although variousassays for detecting Lp-PLA2 protein have been described, such assaystypically describe using only freshly isolated or produced (e.g., withina few minutes, hours or days) Lp-PLA2 to form calibration standards.

To provide meaningful results, quantitative Lp-PLA2 assays need to becalibrated and quality controlled with value assigned reagents. Furtherthe assay needs to measure controls within a predetermined quantitativerange. The controls and calibrators (standards) can be provided within areagent kit, as a separate kit or be acquired as individual valueassigned reagents. Alternatively Lp-PLA2 reagent kit can be calibratedduring manufacturing and calibration values or curves are provided. Inthis case no physical reagent is used by the laboratory running theassay, rather the kit manufacturer uses in-house reagents to generatecalibration curves, values or equivalent for their Lp-PLA2 assay. Forexample, Lp-PLA2 assays include: immunoassays (Caslake, 2000), activityassays (PAF Acetylhydrolase Assay Kit, Cat #760901 product brochure,Cayman Chemical, Ann Arbor, Mich., 12/18/97; Azwell/Alfresa Auto PAF-AHkit available from the Nesco Company, Alfresa, 2-24-3 Sho, Ibaraki,Osaka, Japan or Karlan Chemicals, Cottonwood, Ariz., see also Kosaka(2000)), spectrophotometric assays for serum platelet activating factoracetylhydrolase activity (Clin Chem Acta 296: 151-161, WO 00/32808 (toAzwell)), and other published methods to detect Lp-PLA2 include WO00/032808, WO 03/048172, WO 2005/001416, WO 05/074604, WO 05/113797,U.S. Pat. Nos. 5,981,252 and 5,880,273 and U.S. publication No. US2012-0276569 A1.

As described in greater detail herein, one significant problem,previously not well characterized, with such assays is that thecalibrators, standards and controls have a relatively short “shelf-life”once made, as the Lp-PLA2 within even buffered calibration standardsloses activity and antigenicity after a few months (e.g., beyond 4-6months) to a substantial degree. This effect may be particularly truewhen using recombinant Lp-PLA2. This loss of activity may result in lessaccurate or even erroneous results when attempting to calibrate orquality control an Lp-PLA2 assay. Thus, it would be beneficial toprovide calibration standards and controls, kits including calibrationstandards and controls, assays including calibration standards andcontrols, and methods of making and using them, that include the use ofrecombinant Lp-PLA2 that have long shelf-life and retain stability andactivity for more than 4 months (e.g., for more than 5 months, more than6 months, more than 12 months, more than 18 months, etc.).

SUMMARY OF THE DISCLOSURE

Calibration solutions, standards, or assay controls having predeterminedconcentrations of recombinant Lp-PLA2 are described herein, as well asmethods and kits using them, including methods of calibrating,re-calibrating or confirming results. In particular, described hereinare calibration and control solutions of recombinant Lp-PLA2 (rLp-PLA2)that are stable for an extended period of time (e.g., greater than 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 12 months, 18 months, etc.). The terms calibration standards,calibrator and standard may be used interchangeably in this document. Ingeneral, the solutions described herein are adapted so that therecombinant Lp-PLA2 may have an exceptionally long shelf-life comparedto previously described solutions, and these solution may be directlyused in an assay for determining, calibrating or confirming Lp-PLA2activity and/or concentration. Surprisingly, as described andillustrated below, stability of rLp-PLA2 may be enhanced (even inlow-salt solutions) by including a sufficiently high concentration of adetergent, and particularly a cholate detergent, that it forms micelles.This is surprising in part because common wisdom when describing storageand use of protein samples and standards as part of an assay (such as anELISA-type assay) is to use detergent at relatively low concentrations(and certainly below the critical micelle concentration) so as to avoidpotentially deleterious effects of the detergent on the binding. See,e.g., “inhibition of Protein-Protein Interactions: Non-Cellular AssayFormats” in the Assay Guidance Manual, Arkin, et al. (2012) (“Lowconcentrations of detergents tend to stabilize proteins, reducenonspecific binding of proteins to assay plates, and break up compoundaggregates . . . in general, detergents should be used at concentrationsbelow their critical micelle concentration (CMC).”). The dilution ofconcentrated recombinant enzyme, known as lipoprotein-associatedphospholipase-A2 (Lp-PLA₂), is an integral step in the process ofcreating calibration standards for an in vitro diagnostic assay used todetect the analyte in a clinical setting. Accurate and stablecalibration standards are essential for the requisite traceabilitynecessary when performing clinical in vitro diagnostic assays for theanalyte, such as a PLAC (Lp-PLA2 immunoassay) test. Specific individualdetergents, formulated at concentrations at, or above, their adjustedcritical micelle concentration are required for stabilization andmaintenance of accurate and stable analyte values for the analyte,recombinant Lp-PLA₂ protein, in these buffered calibration standards.Proper and reliable functionality of the calibration standards allow forthe accurate detection of clinical analyte values for Lp-PLA₂ in bodilyfluids, such as blood samples, plasma samples or serum samples. Here,the utility of using specific detergents at appropriate concentrationsat or above their individual salt-adjusted critical micelleconcentrations (CMC's) to stabilize recombinant Lp-PLA₂ in the contextof a calibration standard is demonstrated. Upon dilution, thesedetergents must be utilized at or above critical micelle concentrationin order to protect the enzyme from inactivation, suggesting thatdetergent micelles stabilizing Lp-PLA₂ protein, not individual detergentmolecules (monomers). After the enzyme has been diluted and allowed tobecome inactivated, the subsequent addition of detergents cannot recoverthe enzymatic activity of recombinant Lp-PLA₂.

The dilution of recombinant Lp-PLA₂ enzyme in the absence of certaindetergents results in inactivation of the enzyme via a bifurcatedpathway. Mechanistically, the first route for loss of enzymatic activityin the absence (or, alternatively, at sub-CMC concentrations) ofdetergent is simply an irreversible denaturation of the recombinantprotein due to unfolding. The second route for the loss of enzymaticactivity in the absence (or, alternatively, at sub-CMC concentrations)of detergent is an irreversible self-association of Lp-PLA₂ by theformation of dimers and/or higher order oligomers. Importantly, themonomeric Lp-PLA₂ protein has a propensity to dimers and higher orderoligomers in the absence of certain detergents, certain polar lipidsand/or certain lipoproteins (e.g., binding to HDL or LDL, per itsphysiological context). The presence of specific detergents at or abovetheir salt-adjusted CMC value is essential for preventing both of theseirreversible routes of enzyme inactivation. In particular, thestructurally-related members of the cholate family of detergents,including CHAPS, CHAPSO and sodium (deoxy)cholate have been demonstratedto provide excellent stability in preventing the inactivation of theLp-PLA₂ protein by denaturation and/or formation of higher orderoligomers.

Association of Lp-PLA₂ with Detergent Micelles.

Size exclusion chromatography was performed to estimate the molecularsize of the rLp-PLA₂ the presence and absence of 10 mM CHAPS (CMC=˜6mM). The results indicated that the same enzyme was eluted verydifferently under the various conditions. The expected molecular weightof Lp-PLA₂, not including the glycosylation oligosaccharide chains, isabout 48 kD. To further understand the retention time shift, we resolvedthe enzyme by the same procedure with different detergents. The resultsshowed that the column retained rLp-PLA₂ differently with differentdetergents. Detergents with larger micelle molecular weight elutedrLp-PLA₂ earlier from the column. This indicates the association ofrLp-PLA₂ with the micelles of the detergents. However, the molecularsize of the rLp-PLA₂ in the absence of the detergents seems even largerthan that of the complex containing the enzyme:detergent micelle. Thissuggests that the enzyme forms oligomeric structures or aggregates inthe absence of detergents. In addition, the recovery yield based on theenzymatic activity assay was much lower when rLp-PLA₂ was fractionatedin the absence of detergents. In the absence of detergents, only about23% of rLp-PLA₂ activities were recovered compared to 60-146% recoveryin the presence of detergents. Thus, dilution of rLp-PLA₂ in the absenceof detergents results in irreversible inactivation of the enzyme.

To investigate the lost rLp-PLA₂ in the absence of detergents, purifiedrLp-PLA₂ with a His-tag at the C-terminal was subjected to fractionationand the fractions were assayed by both the CAM assay and the His-ELISAusing rabbit anti-Lp-PLA₂ polyclonal antibody. When rLp-PLA₂ wasfractionated in the absence of detergents, the results indicated thattwo mass peaks (fraction 16-18 and 21-23) were shown by the His-ELISAbut only one activity peak (fraction 16-18) was seen by the CAM assay.That is, the lower molecular weight mass peak (fraction 21-23) containedno enzymatic activity. However, when the enzyme was fractionated in thepresence of 10 mM CHAPS in the same buffer, no mass or enzymaticactivity at fraction 16-18 was seen but both mass and enzymatic activitywere detected at the fraction 21-23. This suggests that the lowermolecular weight peak (fraction 21-23), which probably comes from thehigher molecular weight peak (fraction 16-18), losses its activityirreversibly in the absence of detergents. In the presence ofdetergents, rLp-PLA₂ is probably does not form oligomers, and,furthermore, it is stabilized by the formation of the complexes withdetergent micelles.

Dilution Results in Inactivation of rLp-PLA₂ in the Absence ofDetergents.

Freshly prepared rLp-PLA₂ diluted in the presence or absence ofdetergents had no difference in specific activity when assayed with CAM(results not shown). However, when the enzyme is stored in the absenceof detergents at 4° C. it lost its activity faster, especially at lowanalyte concentrations (results not shown). To further investigate thedecrease of rLp-PLA₂ specific activity in the absence of detergents, theenzyme was subjected to dilution to the final concentration between 1-3μg/ml in PBS, pH 7.2, and the changes of the enzymatic activity andimmuno-reactive mass were followed. The immuno-reactive mass of Lp-PLA₂was quantified by using the PLAC kits that only recognized thenon-denatured form of the enzyme (conformational). The enzyme graduallylost its activity and immuno-reactive mass in two phases. Upon dilution,the enzymatic activity and the immuno-reactive mass had a sharp declinephase (about 1-2 days of incubation at 4° C.) and then the inactivationrate decreased and transferred to a slower phase. The final normalizedlosses in both activity and immuno-reactive mass were in the range of50-75% at the fifteenth day of incubation. Actually, for each reaction,the inactivation rates and final losses of the enzymatic activity andimmuno-reactive mass varied with different experimental conditionsdepending on the final diluted enzyme concentration (see the followingexperiments), the storage conditions of the enzyme, the dilution buffercomponents and incubation temperature, etc.

Detergents have Differential Effects on rLp-PLA₂ Activity.

The effects of detergents on the dilution inactivation of rLp-PLA₂ wereinvestigated. When 10 mM CHAPS was included in the dilution buffer, noinactivation was observed for the diluted rLp-PLA₂ at 1 μg/ml. However,the addition of 10 mM CHAPS into the inactivated enzymes only recovereda very small portion of the lost activity but it did prevent the enzymefrom further inactivation during the extended incubation. In addition toCHAPS, several other non-ionic detergents, such as Tween-20, TritonX-100 and digitonin, were also found protective in the dilutioninactivation of rLp-PLA₂ (data not shown). Detergents with high CMC wereless effective than those with lower CMC. In an experiment of dilutioninactivation for rLp-PLA₂, the diluted enzyme was incubated in bufferscontaining variable detergent concentrations from 0.15 mM to 10 mM. Therate of enzyme inactivation was found to be concentration dependent forCHAPS (CMC=6 mM) and deoxycholate (CMC=1.5 mM) but not for Triton X-100(CMC=0.3 mM), Digitonin (CMC=0.09 mM) and Tween-20 (CMC=0.06 mM). Thissuggests that detergent micelles, possibly instead of or in addition tomonomeric detergent, are the stabilizer of rLp-PLA₂ molecule.

The Effects of the Protein Concentration on the Activity of rLp-PLA₂.

At high concentrations (>0.5 mg/ml), rLp-PLA₂ is fairly stable even inthe absence of detergents (observation not shown). In the dilutioninactivation of the recombinant Lp-PLA₂, the inactivation rates aredependent on the final diluted concentration of the enzyme. Theconcentration effect on the rLp-PLA₂ dilution inactivation isillustrated in. The rate and final loss of the rLp-PLA₂ inactivationupon dilution varied in the enzyme concentration range of 0.6-5 μg/ml.The inactivation rates became relatively independent of final enzymeconcentrations at both ends of the above concentration range. This canbe better demonstrated by plotting the residual residue percentage ofthe rLp-PLA₂ activity after the enzyme was diluted and incubated at 4°C. for ten days against the protein concentrations. In the logisticscale of concentration, it can be fitted into a sigmoidal curve. Thereis a sensitive range between 1 and 5 The saturation at bothconcentration ends may indicate that there is a dynamic equilibriumbetween the stable and unstable forms of rLp-PLA₂, which shiftsdepending on the concentration of the enzyme. Since the inactivation isdue to structural disruption by solvent and irreversible, it should be areaction of first order kinetics, that is, concentration independent.When the enzyme concentration decreases to a certain level, theequilibrium is shifted to the unstable form and then the irreversibleinactivation rate becomes concentration independent. When theconcentration of rLp-PLA₂ increases, the rate of inactivation is reduceddue to the equilibrium shifting to the stable form of the enzyme. Mostlikely, the stable and unstable forms of Lp-PLA₂ should represent theoligomerized and the dissociated enzyme respectively since the dilutionusually causes dissociation and vice versa. At the concentration at 2.5μg/ml or 53 nM, roughly half of rLp-PLA₂ is in monomer and half formsthe aggregate or oligomer as estimated.

Protection of rLp-PLA₂ Activity by Lipoproteins.

Lp-PLA₂ protein has been shown to associate with LDL and HDL in humanplasma (9). Experiments were designed to reveal if LDL and HDL wouldprevent rLp-PLA₂ from the inactivation during the dilution intonon-detergent containing buffers. Purified rLp-PLA₂ was diluted in 50 mMsodium phosphate buffer, pH 7.2, containing 150 mM sodium chloride and 2mM EDTA at the final concentration of 0.5 μg/ml enzyme and incubated at4° C. for 2 days. The experiments were carried out in the presence ofvarious concentrations of fractionated LDL and HDL (devoid of endogenousLp-PLA₂ activity). It was indeed found that the dilution inactivation ofrLp-PLA₂ could be averted in the presence of either LDL or HDLparticles. Human LDL or HDL at concentrations as low as 1.4 and 0.14mg/dL of triglyceride respectively fully protected the rLp-PLA₂ activityduring the dilution in the phosphate buffer. No significant activitylosses were observed after the two day period of incubation at 4° C. inthe LDL or HDL containing buffer while more than 90% of the originalactivity vanished in the control buffer. However, unexpectedly higherconcentrations of LDL or HDL reduced the protection capability possiblydue to the proteolysis of the recombinant enzyme.

The Effects of Chaotropic Agents on the Activity of rLp-PLA₂.

According to the gel permeation experiments, detergents could reduce themolecular weight of rLp-PLA₂ and stabilize its activity. To investigatethe connection between the deoligomerization and stabilization effectsof detergents, rLp-PLA₂ was diluted and incubated at 4° C. in thepresence of 1 M sodium salts of fluoride, bromide, chloride, iodide,nitrate, sulfate (0.5 M) and thiocyanate. While detergents were found tostabilize rLp-PLA₂, anions destabilizing protein-protein interactions,such as SCN⁻¹ or I⁻¹ (22), were found to promote the inactivation of theenzyme. The inactivation of the diluted rLp-PLA₂ during the incubationat 4° C. was significantly accelerated by including 1 M of NaSCN or Nalin the incubation buffer. This is not due to the added sodium saltconcentration because no other salts had effects on the stability of theenzyme. None of the above chemicals (up to 1 M) was found inhibitory tothe enzymatic activity of rLp-PLA₂ either (results not shown). Theexperiment suggests that the protein-protein interaction breaker such asSCN⁻¹ or I⁻¹ actually destabilizes rLp-PLA₂. It may be inferred thatrLp-PLA₂ tenders to form a dimer or oligomers during the incubation but,if the self-interaction is prevented or interrupted by chaotropicagents, the monomeric enzyme will be denatured, possibly due to exposureof the hydrophobic substrate binding site to aqueous solvents.

Chemical Cross-Linking of rLp-PLA₂.

To further confirm the formation of the oligomeric rLp-PLA₂ duringdilution, the highly purified enzyme was diluted into buffers containinga chemical cross-linker, ethylene glycol bis[succinimidylsuccinate](EGS), with and without detergents. In a cross-linking experiment, whenrLp-PLA₂ was diluted to the final concentration of 1 μg/ml in theabsence of detergents, only oligomers with molecular weight >98 kD weredetected on the Western Blot by rabbit anti-Lp-PLA₂ antibody. Nomonomeric (48 kD) and only a low amount of dimeric (98 kD) rLp-PLA₂ wereseen. Second, the extent of rLp-PLA₂ oligomerization observed wasdifferent when stored at different conditions. Enzyme stored in buffercontaining 5 mM CHAPS had a lower oligomerized molecular weight thanenzyme stored in the detergent-free condition although both were dilutedinto the same cross-linking buffer at the same final concentration.Third, in the presence of 10 mM CHAPS (or 1% Tween-20, data not shown),the majority of rLp-PLA₂ stayed monomeric after cross-linked by EGS.Again, the enzyme stored in the presence of 5 mM CHAPS was almost freeof oligomeric bands when cross-linked in buffer containing detergentswhile the detergent-free enzyme still had significant amounts of highmolecular weight species when cross-linked in the same buffer. Theseresults prove that rLp-PLA₂ does quickly self-associate and formpolymers upon dilution in the absence of lipid substrates or detergents.The detergents do not reduce the reactivity of EGS in the cross-linkingof rLp-PLA₂ because the control experiments to internally cross-link IgGby EGS were not altered by the presence of the same detergents (data notshown). Thus, the purified recombinant lipoprotein-associatedphospholipase A2 (rLp-PLA₂) expressed in HEK293 cells has a propensityto form oligomers in the absence of detergents or lipids by chemicalcross-linking.

A Detergent Comparison study was a component swapping experiment inwhich selected membrane detergents/CHAPS analogues were screened in ashort-term real-time stability study. The eleven different detergentvariants in this study included each of those included in the Dojindo“First Choice” detergent screening kit (CHAPS, n-Dodecyl-β-D-maltoside,n-Octyl-β-D-glucoside, sodium cholate and MEGA-8), various CHAPSanalogues (Dojindo detergents CHAPSO, BIGCHAP, deoxy-BIGCHAP), andvarious grades/lot numbers of Sigma CHAPS (including two lots from thecurrent grade of CHAPS used in Manufacturing). All the detergents weresubstituted into the standard calibrator diluent formulation at fourconcentrations each in a linear titration series. The concentrationrange surveyed for each detergent was based on each individualdetergent's published critical micelle concentration (CMC). Mostdetergents were also tested at one concentration above the CMC and twoconcentrations below the CMC, with the single exception being the MEGA-8detergent. The MEGA-8 detergent presented a technical challenge withrespect to testing above its published CMC (58 mM). A key aspect of thisstudy is the detergent concentrations chosen were normalized based ontheir respective critical micelle concentrations (CMC's), a valuespecific to each detergent. With respect to maintaining Lp-PLA₂stability, the general trend for the set of detergents was stabilizedwas maximal when the detergent concentration was at CMC (or higher) witha sharp drop off in stability at concentrations lower than CMC. Thisresult strongly suggests that micelle formation is important formaintaining Lp-PLA₂ stability across the entire panel of detergentssurveyed. When CHAPS was studied to the exclusion of the otherdetergents, the lots of CHAPS analyzed here actually showed slightlybetter stability at sub-CMC concentrations than the other detergents.The 0.595× concentration of CHAPS (corresponding to the standard [4.76mM] CHAPS concentration in the calibrator matrix) showed comparablestability to the 1×CMC concentration, but the stability profile showed˜10% drop-off at the 0.354× concentration (corresponding to the 2.83 mMCHAPS concentration). These results from this thirty-day stability studysuggested that a concentration of CHAPS used in a calibrator diluentformulation (e.g., 4.76 mM) may be close to a “cliff” in CHAPSconcentration with respect to stability performance.

The Detergent Comparison study also demonstrated that there isdifferential calibrator stability observed when using different lots ofCHAPS detergent. In a comparison of four different lots of CHAPS fromtwo vendors, statistically significant differences in stability wereobtained using a Student's t-test even within the timeframe a 30-dayshort-term stability study. Notably, the difference in stability betweenthe Dojindo lot of CHAPS (lot number CT717) and the Sigma lot #3(BioXtra, lot number 18K530041V) yielded a statistically significantdifference at every CHAPS concentration tested. Given that standardconcentrations of the other raw materials were used in this study, theseresults suggest the possibility that differences in stability as afunction of detergent concentration can be observed even in a relativelyshort timeframe. It should be noted, though, that the differential inpercent stability observed with some of these lots of Sigma CHAPS(namely, lots 018K53003 and 040M5319V) is of a greater magnitude thanthat observed in subsequent stability studies with the same two lots ofSigma CHAPS in the Mix-and-Match Study. On the other hand, the singlelot of Dojindo CHAPS tested demonstrated good stability at the standardCHAPS concentration and higher when tested using the same pre-formulatedmaster-mixes of the remaining raw materials common to each formulation.

A variety of other detergents were screened in the Detergent Comparisonstudy to assess the feasibility of using alternate detergents tostabilize Lp-PLA₂. The two CHAPS analogues, CHAPSO and sodium cholate,showed promising short-term stability results. The n-octyl-b-glucosideshowed some promise with its performance in this initial screen; thisdetergent was used in the determination of the structure of Lp-PLA₂ byx-ray crystallography (Samanta 2008). The n-octyl-b-maltoside showedless promising short term stability indicated by a sharp drop-off inpercent stability between the Day 14 time point and the Day 0 timepoint. The MEGA-8 may require a relatively high detergent concentration(˜30 mM) for effective protein stabilization.

In general, described herein are calibration solutions having a verylong shelf-life. As used herein, shelf-life refers to the time duringwhich the solution stably maintain a predetermined level of functional,properly-folded lipoprotein-associated phospholipase A2 (Lp-PLA₂). Thusthe shelf-life may refer to the length of time during which apredetermined amount (e.g., more than 95%, more than 90%, more than 85%,more than 80%, etc.) of the concentration and/or activity of the Lp-PLA2within the solution is retained.

As described in greater detail below for the first time, a calibrationsolution of recombinant Lp-PLA2 having a long shelf-life may include apredetermined amount of Lp-PLA2 (e.g., predetermined dilution) in abuffer solution that includes sufficient micelles to stabilize therecombinant Lp-PLA2. The micelles may be made of a cholat detergent(e.g., CHAPS, CHAPSO, sodium (deoxy)cholate, etc.) at a concentrationabove the critical micelle concentration (CMC), as well as apreservative, salt (e.g., non-chaotropic salt), pH buffer and proteinbuffered matrix.

For example, described herein are lipoprotein-associated phospholipaseA2 (Lp-PLA2) calibrator kits for use with an Lp-PLA2 assay, the kithaving a shelf-life of greater than 4 months (e.g., greater than 5months, greater than 6 months, greater than 7 months, greater than 8months, greater than 9 months, greater than 10 months, greater than 11months, greater than 12 months, greater than 13 months, greater than 14months, greater than 15 months, greater than 16 months, greater than 17months, etc.). A kit may include: a first calibration solutioncomprising a first concentration of a recombinant Lp-PLA2 in a firstbuffer solution, wherein the first buffer solution comprises a pluralityof micelles of a first detergent stabilizing the recombinant Lp-PLA2;and a second calibration solution comprising a, second concentration ofthe recombinant Lp-PLA2 in a second buffer solution, wherein the secondbuffer solution comprises a plurality of micelles of a second detergentstabilizing the recombinant Lp-PLA2.

Any of calibrator kits (which may be separate from or included as partof an assay for identifying Lp-PLA2), may include a plurality ofcalibration solutions, where each solution has a predetermined amount ofrecombinant Lp-PLA2. For example, a kit may include at least threecalibration solutions each having a different but known concentration ofLp-PLA2 in a buffer solution, wherein the buffer solution comprisesmicelles of a detergent; the micelles act to stabilize the recombinantLp-PLA2.

In general, the primary detergent (forming the micelles in the buffer)may be any appropriate detergent, including (but not limited to) membersof the cholate family of detergents. The concentration of primarydetergent is generally above the CMC. Although different buffersolutions for the different calibration concentrations may be used, ingeneral the same calibration buffer compositions may be used, with theexception of the differing concentrations of recombinant Lp-PLA2. Forexample, the first and second primary detergent may comprise CHAPS(e.g., at a concentration that is above the CMC for the amount of saltin the buffer solution).

In any of the variations described herein, the calibration buffersolutions including the recombinant Lp-PLA2 may be “low salt” (e.g.,less than 1 M salt concentration) buffer solutions. As described ingreater detail below, such low-salt solutions may include a seconddetergent (e.g., a surfactant such as TWEEN 80) to prevent aggregationof the recombinant Lp-PLA2, in addition to the detergent forming themicelles. Any of the buffer solutions described herein may include asalt that is a non-chaotropic salt. For example, the buffer solution mayinclude comprises one or more of: NaCl and an acetate salt. Any of thecalibration solutions described herein may also include a preservative(e.g., sodium azide).

The buffer solutions described herein may also typically include aprotein buffered matrix, such as a bovine serum albumin (BSA). Any ofthe calibration buffer solutions described herein may also include a pHbuffer (e.g., Tris).

For example, a lipoprotein-associated phospholipase A2 (Lp-PLA2)calibrator kit for use with an Lp-PLA2 assay, having a shelf-life ofgreater than 4 months, may include: a first calibration solutioncomprising a first concentration of a recombinant Lp-PLA2 in a firstbuffer solution, wherein the first buffer solution comprises a pluralityof micelles of a cholate detergent stabilizing the recombinant Lp-PLA2,a protein buffered matrix, a pH buffer and a preservative; and a secondcalibration solution comprising a second concentration of therecombinant Lp-PLA2 in a second buffer solution, wherein the secondbuffer solution comprises a plurality of micelles of the cholatedetergent stabilizing the recombinant Lp-PLA2.

Also described herein are lipoprotein-associated phospholipase A2(Lp-PLA2) assays having recombinant calibrators, the assay comprising: aplurality of calibrator solutions each comprising a predeterminedconcentration of a recombinant Lp-PLA2 in a buffer solution, wherein thebuffer solution comprises a plurality of micelles of a cholate detergentstabilizing the recombinant Lp-PLA2; a wash buffer; a solid phasesupport configured to bind Lp-PLA2; and a report antibody specific toLp-PLA2. The calibrator solutions may include any of the calibratorbuffer solutions described herein, including in particular a cholatedetergent is above a critical micelle concentration (CMC) for thedetergent. Although this example describes an immunoassay kit (e.g., amass kit) other Lp-PLA2 assays may be based on activity (enzymaticactivity) and may include a substrate and detection means (e.g.,colorimetric, radioactive, etc.) along with the calibration standards.

A lipoprotein-associated phospholipase A2 (Lp-PLA2) assay havingrecombinant value-assigned solutions having a shelf-life of more than 4months, the assay may comprise: a plurality of value-assigned solutionseach comprising a predetermined concentration of a recombinant Lp-PLA2in a low-salt buffer solution having a salt concentration below about 1M, wherein the buffer solution comprises a cholate detergent forming aplurality of micelles that stabilizes the recombinant Lp-PLA2; asolution comprising an agent that interacts with Lp-PLA2 to produce adetectable signal; and a wash buffer.

Also describe are lipoprotein-associated phospholipase A2 (Lp-PLA2)assay utilizing a value-assigned solution having a long shelf-life foruse as a standard, control, calibrator or re-calibrator, may include: avalue-assigned solution comprising a predetermined concentration of arecombinant Lp-PLA2 in a buffer solution, wherein the buffer solutioncomprises a cholate detergent forming a plurality of micelles thatstabilize the recombinant Lp-PLA2; a wash buffer; a solid phase supportconfigured to bind Lp-PLA2; and a report antibody specific to Lp-PLA2.

As mentioned, low-salt calibration solutions may be used. In general, alow salt calibration solution includes less than 1 M salt in addition tomicelles that help stabilize the recombinant Lp-PLA2, and may alsoinclude a secondary detergent (e.g., a surfactant such as Tween-20).

For example, a lipoprotein-associated phospholipase A2 (Lp-PLA2)calibrator kit for use with an Lp-PLA2 assay having a shelf-life ofgreater than 4 months may include: a first calibration solutioncomprising a first concentration of a recombinant Lp-PLA2 in a firstlow-salt buffer solution having a salt concentration below about 1 M,wherein the first buffer solution comprises a plurality of micelles of afirst detergent stabilizing the recombinant Lp-PLA2 and a firstsecondary detergent to prevent aggregation of the recombinant Lp-PLA2;and a second calibration solution comprising a second concentration ofthe recombinant Lp-PLA2 in a second low-salt buffer solution having asalt concentration below about 1 M, wherein the second low-salt buffersolution comprises a plurality of micelles of a second detergentstabilizing the recombinant Lp-PLA2 and a second secondary detergent toprevent aggregation of the recombinant Lp-PLA2.

For example, a lipoprotein-associated phospholipase A2 (Lp-PLA2)calibrator kit for use with an Lp-PLA2 assay, having a shelf-life ofgreater than 4 months, may include: a first calibration solutioncomprising a first concentration of a recombinant Lp-PLA2 in a firstlow-salt buffer solution having a salt concentration below about 1 M,wherein the first buffer solution comprises a plurality of micelles of acholate detergent stabilizing the recombinant Lp-PLA2, a first secondarydetergent to prevent aggregation of the recombinant Lp-PLA2, a proteinbuffered matrix, a pH buffer and a preservative; and a secondcalibration solution comprising a second concentration of therecombinant Lp-PLA2 in a second low-salt buffer solution having a saltconcentration below about 1 M, wherein the second buffer solutioncomprises a plurality of micelles of the cholate detergent stabilizingthe recombinant Lp-PLA2 and a second secondary detergent to preventaggregation of the recombinant Lp-PLA2.

Also described herein are methods of calibrating and methods ofrecalibrating an assay for Lp-PLA2. For example, described herein aremethods of recalibrating a calibration curve for detection oflipoprotein-associated phospholipase A2 (Lp-PLA2) from a biologicalsample using a value-assigned solution of recombinant Lp-PLA2 having along shelf life. The method for recalibration may include: detecting afirst signal from a value-assigned solution having a first predeterminedconcentration of a recombinant Lp-PLA2 in a buffer solution, wherein thebuffer solution comprises a detergent forming a plurality of micellesthat stabilize the recombinant Lp-PLA2; and transforming the calibrationcurve using the first signal.

In general, a method of recalibrating may be used to adjust apredetermined calibration curve. The predetermined calibration curve maybe factory or lot defined and may be provided with a kit or collectionof reagent used to quantify the level and/or activity of Lp-PLA2.Recalibration generally involves taking one or more measurements(signals) from value-assigned solutions of rLp-PLA2. A value-assignedsolution is one in which the amount of rLp-PLA2 is known or set to apredetermined level. Thus, a value-assigned solution may be a standard,a calibration solution, etc.

Any of the recalibration methods described herein may include a step oftransforming (e.g., adjusting) a preexisting calibration curve based onthe signal(s) collected from one or more value-assigned solutions.Transforming may include shifting, scaling or shifting and scaling thecalibration curve based on the first signal. The step of transforming istypically performed by a machine such as a computer (processor) or thelike, which may be configured (specifically configured by includingsoftware, hardware or firmware) that adjusts an initial calibrationcurve based on the detected signal(s) from the value-assigned(‘recalibration’) solutions. The known value (e.g., concentration value,activity, etc.) of the value-assigned solution may be correlated withthe detected signal. In some variations the calibration curve may be fit(e.g., suing the machine) to the signals measured for the value-assignedsolution(s).

Any appropriate calibration curve may be used. For example, acalibration curve may show a relationship between signal (e.g., measuredas optical signal, etc.) and concentration of Lp-PLA2 and/or activity ofLp-PLA2. For example, in some variations the calibration curve relatessignal intensity to concentration of Lp-PLA2.

Any number of value-assigned solutions may be used to providecalibration/re-calibration signals. For example, the method may includedetecting a second (or more) signal from a second value-assignedsolution having a second predetermined concentration of a recombinantLp-PLA2 in the buffer solution, wherein the buffer solution comprisesthe detergent forming a plurality of micelles that stabilize therecombinant Lp-PLA2, and wherein transforming the calibration curvecomprises using the first and second signals.

In general, the methods of calibration and recalibration (as well asmethods for normalizing or performing a control) of Lp-PLA2 assaysdescribed herein may include combining the value-assigned solution(s)with an agent that interacts with Lp-PLA2 to produce a detectable signalbefore detecting the first signal. Any appropriate agent may be used,including an agent that interacts with Lp-PLA2 to form a detectablecomplex, such as an antibody directed against Lp-PLA2 or a substrate forLp-PLA2. Alternatively, the agent may be or may include a substrate onwhich the Lp-PLA2 acts. For example, the agent that interacts withLp-PLA2 may comprise a labeled antibody directed against Lp-PLA2 or alabeled substrate for Lp-PLA2.

Detecting signal may include detecting a complex of Lp-PLA2 and anantibody or detecting enzymatic activity Lp-PLA2.

As described in greater detail herein, value-assigned solutions ofrLp-PLA2 that are of particular interest and utility include thosehaving a plurality of micelles of a detergent. Thus, the detergent (afirst detergent) may be above the critical micelle concentration (CMC)for the detergent in the context of the value-assigned solution. Thedetergent may be, in particular a cholate detergent (e.g., CHAPS, etc.).In addition to the micelles of detergent that stabilize the rLp-PLA2,the solutions described herein may also include additional detergent(the same detergent or a different detergent) that prevents aggregationof the Lp-PLA2. Any of the buffer solutions in which the rLp-PLA2 arepresent may be configured as low-salt solutions (e.g., having less than1 M total salt). For example, detecting signal from the value-assignedsolutions may include detecting the first signal from the value-assignedsolution having the first predetermined concentration of a recombinantLp-PLA2 in the buffer solution, wherein the buffer solution is alow-salt buffer solution having a salt concentration below about 1 M andcomprising a detergent forming the plurality of micelles and a seconddetergent to prevent aggregation of the recombinant Lp-PLA2, furtherwherein the detergent forming the plurality of micelles is differentfrom the second detergent.

Detecting signal may include detecting the first signal from thevalue-assigned solution having the first predetermined concentration ofa recombinant Lp-PLA2 in the buffer solution, wherein the buffersolution further comprises a protein buffered matrix (e.g., PBS). Asdescribed herein, the type and concentration of the protein bufferedmatrix (or any of the other components of the solution) may be chosen tooptimize the shelf-life.

For example, described herein are methods of recalibrating a calibrationcurve for detection of lipoprotein-associated phospholipase A2 (Lp-PLA2)from a biological sample using a value-assigned solution of recombinantLp-PLA2 having a long shelf life, the method comprising: combining avalue-assigned solution comprising a first predetermined concentrationof a recombinant Lp-PLA2 in a buffer solution with an agent thatinteracts with Lp-PLA2 to produce a detectable first signal, wherein thebuffer solution comprises a detergent forming a plurality of micellesthat stabilize the recombinant Lp-PLA2; detecting the first signal; andtransforming a calibration curve by shifting, scaling or shifting andscaling the calibration curve based on the first signal.

In addition to method of re-calibrating a calibration cure, alsodescribed herein are methods of producing calibration curves (fordetection of lipoprotein-associated phospholipase A2 (Lp-PLA2) from abiological sample) using the value-assigned solutions of recombinantLp-PLA2 that have a long shelf life described herein. For example, amethod of generating a calibration curve may include: combining an agentthat interacts with Lp-PLA2 to produce a detectable signal with aplurality of value-assigned solutions, wherein each value-assignedsolution has a predetermined concentration of the recombinant Lp-PLA2 ina buffer solution, the buffer solution comprising a detergent forming aplurality of micelles that stabilize the recombinant Lp-PLA2; detectingLp-PLA2 signals from the value-assigned solutions; and creating acalibration curved based on the relationship between the detectedsignals and the predetermined concentrations of the recombinant Lp-PLA2.

In general, detecting Lp-PLA2 signals from the value-assigned solutionsmay include detecting Lp-PLA2 signals from at least four value-assignedsolutions having different predetermined concentrations of therecombinant Lp-PLA2 (e.g., more than four, more than five, more thansix, more than seven, more than eight, more than nine, more than ten,etc.). For example, detecting Lp-PLA2 signals may comprise detectingLp-PLA2 signals from between about four to 10 value-assigned solutionshaving different predetermined concentrations of the recombinantLp-PLA2.

Since the methods of detecting Lp-PLA2 activity/amount, method ofre-calibrating a calibration curve and methods of generating acalibration curve described herein are specifically for use with theimproved solutions (e.g., value-assigned solutions having a longshelf-life) described, the outcome of such methods may be significantlydifferent from other methods that do not use these solutions. Forexample, although prior art methods for detecting concentration and/oractivity of Lp-PLA2 are performed in a low-detergent buffer (e.g., belowthe CMC), surprisingly the inventors have herein found that assayingactivity and/or binding of Lp-PLA2 in the presence of micelles, as wellas storing rLp-PLA2 in the presence of micelles, has a beneficialeffect. Thus, any of the detection steps for detecting activity and/orbinding of Lp-PLA2 may be performed in the presence of micelles ofdetergent, and/or in a low-salt buffer.

In addition any of the methods described herein may be performed with avalue-assigned solution that has been stored for more than four months(e.g., more than five months, more than six months, etc.).

As mentioned, a calibration curve may relate a signal intensity of thesignals to the predetermined concentrations of the recombinationLp-PLA2.

In any of the methods described herein, signal may be detected bycombining the rLp-PLA2 (or for determining an unknown, a sample from apatient including Lp-PLA2) with an agent generates a detectable signal.The signal may be directly or indirectly detected. For example, theagent may be an antibody that binds or complexes with the rLp-PLA2 (orLp-PLA2) such as an antibody directed against Lp-PLA2 or a substrate forLp-PLA2. The agent that interacts with Lp-PLA2 may be, for example, alabeled antibody directed against Lp-PLA2 or a labeled substrate forLp-PLA2. The label may be optically detectable (e.g., florescent, HRP,etc.) or it may be radio detectable, or the like. In some variations thesignal is indirectly detectable as, for example, when the enzymaticactivity of the rLp-PLA2 (or endogenous Lp-PLA2) is detectable bydetecting a product resulting from the enzymatic activity of theLp-PLA2.

Detecting LpPLA2 signals may comprise detecting a complex of Lp-PLA2 andan antibody or detecting enzymatic activity Lp-PLA2 on a substrate aftercombining the solution including rLp-PLA2 with an agent to generate adetectable signal. Combining may comprise combining the agent with eachof the plurality of value-assigned solutions, wherein the buffersolution of the value-assigned solutions comprises a plurality ofmicelles of CHAPS that stabilize the recombinant Lp-PLA2.

The step of combining may comprise combining the agent with each of theplurality of value-assigned solutions, wherein the buffer solution ofthe value-assigned solutions comprises a low-salt buffer solution havinga salt concentration below about 1 M and a second detergent to preventaggregation of the recombinant Lp-PLA2, further wherein the detergentforming the plurality of micelles that stabilize the recombinant Lp-PLA2is different from the second detergent.

In some variations, the step of combining may comprise combining theagent with each of the plurality of value-assigned solutions, whereinthe buffer solution of the value-assigned solutions comprises a proteinbuffered matrix.

In general, creating a calibration curve may include arranging thesignals from the value-assigned solutions versus the predeterminedconcentrations of the recombinant Lp-PLA2 in the value-assignedsolutions. A curve may be fit to the resulting arrangement. The curvemay be first order, second order, third order, etc. A mathematicalexpression for the curve may be provided (e.g., by the apparatus, e.g.,software, firmware, hardware), and this mathematical expression may beused to determine an estimate of the value of Lp-PLA2 concentrationand/or activity from a biological sample.

For example, a method of producing a calibration curve for detection oflipoprotein-associated phospholipase A2 (Lp-PLA2) from a biologicalsample by using value-assigned solutions of recombinant Lp-PLA2 thathave a long shelf life may include: combining an agent that interactswith Lp-PLA2 to produce a detectable signal with a plurality ofvalue-assigned solutions, wherein each value-assigned solution has adifferent predetermined concentration of the recombinant Lp-PLA2 in abuffer solution, the buffer solution comprising a detergent forming aplurality of micelles that stabilize the recombinant Lp-PLA2, a pHbuffer, a protein buffered matrix and a non-chaotropic salt; detectingLp-PLA2 signals from the value-assigned solutions; and creating acalibration curved based on the relationship between the detectedsignals and the predetermined concentrations of the recombinant Lp-PLA2.

As mentioned above, kits are also described herein. Any of the solutions(including value-assigned solutions of rLp-PLA2) may be included as partof a kit or set. In general a kit may be pre-assembled so that a user isprovided with all of the component parts (e.g., in a single container,or connected container) or it may be assembled by the user fromdifferent or separately provided components. In general, the kitincludes multiple different items that may be used as described herein.

For example, a lipoprotein-associated phospholipase A2 (Lp-PLA2) kit foruse with an Lp-PLA2 assay, the kit having a shelf-life of greater than 4months, may include: a first value-assigned solution comprising a firstpredetermined concentration of a recombinant Lp-PLA2 in a first buffersolution, wherein the first buffer solution comprises a first detergentforming a plurality of micelles that stabilize the recombinant Lp-PLA2;and a second value-assigned solution comprising a second predeterminedconcentration of the recombinant Lp-PLA2 in a second buffer solution,wherein the second buffer solution comprises a second detergent formingplurality of micelles that stabilize the recombinant Lp-PLA2. The kitmay also include a third (or fourth, fifth, sixth, etc.) value-assignedsolution comprising a third predetermined concentration of therecombinant Lp-PLA2 in a third buffer solution, wherein the third buffersolution comprises a third detergent forming a plurality of micellesthat stabilize the recombinant Lp-PLA2. The first and second detergents(e.g., the detergents forming the micelles) may comprise a cholatedetergent, such as CHAPS, at greater than the CMC for the buffer. Ingeneral, the first and second buffer solutions may be the same solution.In particular, the first buffer solution and the second buffer solutionmay be a low-salt buffer solution (e.g., having a total saltconcentration of less than 1 M). The first buffer solution and thesecond buffer solution comprises a low-salt buffer solution comprising anon-chaotropic salt. The first buffer solution and the second buffersolution may comprise a low-salt buffer solution comprising one or moreof: NaCl and an acetate salt. The first buffer solution and the secondbuffer solution may comprise a protein buffered matrix, e.g., bovineserum albumin (BSA). The first buffer solution and the second buffersolution may include Tris as a pH buffer.

In some variations the kit includes a ‘blank’ that includes the bufferwithout any rLp-PLA2. For example, the second predeterminedconcentration of the recombinant Lp-PLA2 of the kit may be zero.

A lipoprotein-associated phospholipase A2 (Lp-PLA2) kit for use with anLp-PLA2 assay, the kit having a shelf-life of greater than 4 months, mayinclude: a first value-assigned solution comprising a firstpredetermined concentration of a recombinant Lp-PLA2 in a first buffersolution, wherein the first buffer solution comprises a cholatedetergent forming a plurality of micelles that stabilize the recombinantLp-PLA2, a protein buffered matrix (e.g., BSA), a pH buffer and apreservative; and a second value-assigned solution comprising a secondpredetermined concentration of the recombinant Lp-PLA2 in a secondbuffer solution, wherein the second buffer solution comprises a cholatedetergent forming a plurality of micelles that stabilize the recombinantLp-PLA2. For example, the cholate detergent of the first and secondbuffer solution may comprise CHAPS. The preservative of the first andsecond buffer solution may comprise sodium azide.

Also described are methods of estimating the amount, activity or amountand activity of lipoprotein-associated phospholipase A2 (Lp-PLA2) from apatient sample, the method comprising: combining at least avalue-assigned solution comprising a first predetermined concentrationof a recombinant Lp-PLA2 in a buffer solution with an agent thatinteracts with Lp-PLA2 to produce a detectable first signal, wherein thebuffer solution comprises a detergent forming a plurality of micellesthat stabilize the recombinant Lp-PLA2; detecting the first signal;combining at least a portion of the patient sample with the agent thatinteracts with Lp-PLA2 to produce a detectable second signal; detectingthe second signal; and assigning a value for activity, concentration oractivity and concentration of Lp-PLA2 from the patient sample using thesecond signal. Assigning the value for an activity, concentration oractivity and concentration of Lp-PLA2 from the patient sample mayinclude calibrating the second signal based on the first signal. Thesemethods may also include determining the validity of the assigned valueby comparing the value of the first signal to a predetermined value or apredetermined range of values.

In some variations, the method may include combining a secondvalue-assigned solution comprising a second predetermined concentrationof a recombinant Lp-PLA2 in a second buffer solution with the agent thatinteracts with Lp-PLA2 to produce a detectable third signal, wherein thesecond buffer solution comprises a plurality of micelles of a detergentstabilizing the recombinant Lp-PLA2; and detecting the third signal.

In general, combining the value-assigned solution with the solutioncomprising the agent that interacts with Lp-PLA2 may include using avalue-assigned solution that has a shelf-life of greater than 4 months.As mentioned above, the agent may be an antibody that binds to Lp-PLA2(including a labeled antibody, e.g., conjugated to an indicator).Combining the value-assigned solution with the solution comprising theagent that interacts with Lp-PLA2 may comprise combining thevalue-assigned solution with the solution comprising a substrate toLp-PLA2.

Any of the value-assigned solutions (e.g., calibrators, standards,controls, etc.) having rLp-PLA2 described herein may be specificallyconfigured as a low-salt solution that has a long shelf-life. Suchsolutions may include a first detergent forming a plurality of micellesstabilizing the rLp-PLA2 (e.g., where the detergent has a concentrationthat is above the CMC), and a second detergent that prevents aggregationof the rLp-PLA2. The first and second detergents may be different (e.g.,a cholate detergent and a polysorbate detergent) or, in some variationsthey may be the same detergent. For example, the detergent forming themicelles may be sufficient (e.g., at a sufficient concentration) to bothform micelles and to separately prevent aggregation of the rLp-PLA2.

For example, a value-assigned solution of lipoprotein-associatedphospholipase A2 (Lp-PLA2) for use with an Lp-PLA2 assay as a control,standard, calibrator or re-calibrator, the value-assigned solutionhaving a shelf-life of greater than 4 months, may include: a firstpredetermined concentration of a recombinant Lp-PLA2 in a low-saltbuffer solution having a salt concentration below about 1 M, wherein thelow-salt buffer solution comprises a detergent (e.g., a cholatedetergent such as CHAPS) forming a plurality of micelles that stabilizethe recombinant Lp-PLA2. The value-assigned solution may include asecond detergent to prevent aggregation of the recombinant Lp-PLA2. Thesecond detergent comprises a non-ionic detergent, such as a polysorbatedetergent (e.g., Tween 80, Tween-20, etc.). The salt in the low-saltbuffer solution typically comprises a non-chaotropic salt (e.g., NaCland an acetate salt).

For example, a value-assigned solution of lipoprotein-associatedphospholipase A2 (Lp-PLA2) for use with an Lp-PLA2 assay as a control,standard, calibrator or re-calibrator, the value-assigned solutionhaving a shelf-life of greater than 4 months, the value-assignedsolution comprising: a first predetermined concentration of arecombinant Lp-PLA2 in a low-salt buffer solution having a saltconcentration below about 1 M, wherein the low-salt buffer solutioncomprises a detergent forming a plurality of micelles that stabilize therecombinant Lp-PLA2 and a second detergent to prevent aggregation of therecombinant Lp-PLA2.

Also described herein are lipoprotein-associated phospholipase A2(Lp-PLA2) kits for use with an Lp-PLA2 assay, the kit having ashelf-life of greater than 4 months, the kit comprising: a firstvalue-assigned solution comprising a first predetermined concentrationof a recombinant Lp-PLA2 in a first low-salt buffer solution having asalt concentration below about 1 M, wherein the first buffer solutioncomprises a cholate detergent forming a plurality of micelles thatstabilizes the recombinant Lp-PLA2, a protein buffered matrix, a pHbuffer and a preservative; and a second value-assigned solutioncomprising a second low-salt buffer solution having a salt concentrationbelow about 1 M, wherein the second buffer solution comprises aplurality of micelles of the cholate detergent (e.g., CHAPS).

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe claims that follow. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a table showing retention time and recovery yield ofrecombinant Lp-PLA2 (rLp-PLA2). Purified rLp-PLA2 was fractionated usinga newly purchased Superose-6 column and 5-25 μl of each fraction wereassayed by CAM as described in the experimental section. Peak fractionswere used to represent the retention time. Yields were calculated viadividing the total activity units from all fractions by the totalactivity units injected. Over recovery of Triton X-100 was due to thehigh background of the detergent in CAM assay.

FIGS. 1A-1C shows fractionation of rLp-PLA2 with a Superose-6 column.FIG. 1A shows Fractionation of rLp-PLA2 with Superose-6 column in thepresence and absence of 10 mM CHAPS. Ten uL of purified rLp-PLA2 in 50mM Tris/HCL, pH 8.0, containing 5 mM CHAPS at the concentration of 1.1mg/ml were fractionated by a 10 cm×300 cm Sperose-6 column equilibratedin 50 mM sodium phosphate, pH 7.4 containing 100 mM sodium chloride, 2mM EDTA and 0.02% sodium azide. Fractions were collected at 0.6 ml/tubewith 0.3 mm/min flow rate. Five uL from each fraction were assayed CAMas described. FIG. 1B shows Fractionation of C-terminal His-tag rLp-PLA2with Superose-6 column in the absence of detergents. Eleven uL ofpurified rLp-PLA2 with a C-terminal His-tag at 2.96 mg/ml werefractionated in the same process as in A. Forty five uL from eachcollected fraction were assayed by CAM and fifty uL were assayed byHisGrap-ELISA. FIG. 1C shows fractionation of purified C-terminalHIS-tag rLp-PLA2 with Superose-6 column in the presence of 10 mM CHAPS.Fifteen uL of rLp-PLA2 with a C-terminal His-tag at 0.7 mg/ml werefractionated in the same process as in IA. Fifty uL from each collectedfraction were assayed by CAM and fifty uL were assayed by HisGrap-ELISA.

FIG. 2 shows a graph of the normalized activity versus time ofincubation. Purified rLp-PLA2 was diluted into PBS, pH 7.54, at thefinal concentration of 2.9 ug/ml and the solution was incubated at 4° C.Five uL of the solution was drawn at the indicated time for the activityassay by CAM. One uL was further diluted 50 fold and 20 uL of thediluted solution were assayed for mass by PLAC. All mass and activityvalues were normalized against the initial value.

FIGS. 3A and 3B show the effects of detergents on the dilution inactivation and activity of rLp-PLA2. FIG. 3A is a graph showingprotection of rLp-PLA2 from dilution inactivation by 10 mM CHAPS.Purified rLp-PLA2 in 50 mM Tris/HCl, pH 8.0 with 10 mM CHAPS was diluted1000 fold into PBS, pH 7.5, with (▾) and without (▪) 10 mM CHAPS at thefin al concentration of 1.3 ug/ml. Samples were incubated at 4° C. Atthe indicated time point, 5 uL of each sample were used to assay foractivity by CAM as described. At the 15th day of the incubation, 100 uLof the enzyme mixture without detergent were withdrawn and mixed with 2uL of 0.5 M CHAPS to obtain the final detergent concentration of 10 mM(▴). Enzymatic activities were monitored for another 10 days using thesame method. FIG. 3B is a graph showing a dose dependence of detergentsin the protection of the rLp-PLA2 from dilution in activation. PurifiedrLp-PLA2 in 50 mM Tris/HCl, pH 8.0 with 10 mM CHAPS was diluted 1340fold at the final concentration of 1 ug/ml into 50 mM sodium phosphate,pH 7.0, containing 100 mM sodium chloride and various concentration ofdetergents: CHAPS (▪), sodium deoxycholate (▴), triton X-100 (▾),Digitonin (♦) and Tween-20 (). The mixtures were incubated at roomtemperature and the activities of the enzyme were assayed by CAM asdescribed in the experimental section. The initial inactivation rateswere obtained by linear regression analyses of the rLp-PLA2 inactivationwithin the incubation time from 0 to 500 minutes and plotted against thelogistic values of detergent concentrations, are presented as aLineweaver-Burke plot.

FIGS. 4A and 4B graphically show activity of the rLp-PLA2 over time ofincubation and residual activity per pLp-PLA2 concentration, showingconcentration effects on the dilution inactivation of rLp-PLA2. In FIG.4A, the rLp-PLA2 in 50 mM Tris/HCL, pH 8.0, with 10 mM CHAPS was dilutedinto PBS, pH 7.2 at the indicated final concentrations. Activities werefollowed by CAM assay at the indicated time. Volumes used for assayswere adjusted based on the concentration of the enzymes so that thedetermined activities were in the linear range. Activities werenormalized to the initial values. Assay conditions were as described inthe experimental section. FIG. 4B shows normalized activities on Day 10,plotted against the final concentrations of rLp-PLA2.

FIG. 5 graphically illustrates protection of rLp-PLA2 from inactivationduring dilution by HDL and LDL. Purified rLp-PLA2 was diluted in 50 mMsodium phosphate buffer, pH 7.2, containing 150 mM sodium chloride and 2mM EDTA at the final concentration of 0.5 ug/ml enzyme and incubated at4° C. for 2 days. The experiments were carried out in the presence ofvarious concentrations of fractionated LDL and HDL (devoid of endogenousLp-PLA2 activity). Only the selected data are presented and thelipoprotein concentrations are indicates as that of triglyceride.

FIG. 6 illustrates the effects of chaotropic agents on the stability andactivity of rLp-PLA2. The effects of different anions on the dilutioninactivation of rLp-PLA2 are shown. Purified rLp-PLA2 in 50 mM Tris, pH8.0, with 10 mM CHAPS was diluted 1000 fold to the final concentrationof 1.3 ug/ml in 12.5 mM sodium phosphate, pH 7.6, containing 1 M of theindicated salt. The enzyme mixtures were incubated at 4° C. and theactivities were followed by CAM assay as described, using 5 uL of theenzyme mixture. Data points were fitted with Boltzmann sigmoidal curves.

FIG. 7 shows cross-linking of rLp-PLA2. rLp-PLA2 was stored andcross-linked under different conditions as indicated. The cross-linkedproteins were then resolved by SDS-PAGE and signals were detected byWestern analyses.

FIGS. 8A-8D illustrate a detergent Comparison Study: CalibratorStability, Full Panel of Detergents. The full panel of detergents usedfor formulate the test calibrators is shown on the Variability Chart(8A) with percent stability shown as a function of detergent identity,day of study (time point, in days), and detergent concentration relativeto CMC (i.e., magnitude of the variation due to the differentformulations) using the JMP 9.0.2 statistical software package. Thereare forty-four detergent combinations used in the formulated calibratorsused in this study that vary by identity and concentrations. Inaddition, four lots of the standard CHAPS detergent were sourced fromtwo different vendors (Sigma and Dojindo) and of two different grades(Sigma standard grade and BioXtra grade) were surveyed. The detergentsand their published CMC values (Dojindo) are as follows: BIGCHAP (CMC:2.9 mM), CHAPS (CMC: 8 mM), CHAPSO (CMC: 8 mM), deoxy-BIGCHAP (CMC: 1.4mM), MEGA-8 (CMC: 58 mM), n-Dodecyl-β-D-maltoside (CMC: 0.17 mM),n-octyl-β-glucoside (CMC: 25 mM), sodium cholate (CMC: 14 mM). Exceptfor the MEGA-8 detergent (for technical reasons related to its extremelyhigh CMC value), the 1.00 concentration represents thedetergent-specific CMC for all the other detergents surveyed. For theMEGA-8 detergent, the 1.00 concentration represents a finalconcentration of 34.51 mM, and the 1.68 concentration represents a finalconcentration of 50.00 mM. Percent stability of formulated calibratorsis calculated relative to refrigerated kit calibrators. As shown in thelower right box, the green hatched line indicates target of 100%stability, and red dashed lines indicate the provisional 97%-103%calibrator stability specification used throughout these studies. Groupmeans are shown according the legend on the right. Gauge analysistrending for percent stability is shown for detergent identity (8B), dayof study (8C), and detergent concentration relative to CMC (8D). Trendline indicates the mean response by each main effect in the model. Theprovisional calibrator stability specification of 100%+/−3% is asdescribed in FIG. 8A.

FIGS. 9A-9D. Detergent Comparison Study: Calibrator Precision, FullPanel of Detergents. (9A) The full panel of detergents used to formulatethe test calibrators is shown on the Variability Chart with precisionshown as a function of detergent identity, day of study (time point, indays), and detergent concentration relative to CMC. The general formatof the Variability Chart format is as described in FIG. 8A. The grandmean of the coefficient of variation (% CV) for all samples in thisstudy was 1.81% and is shown as a dashed line. A gray hatched line showsthe target % CV of 0.00%. A lower % CV value is superior to a higherone. Group means are shown according the legend on the right. Gaugeanalysis trending for precision is shown for detergent identity (9B),day of study (9C), and detergent concentration relative to CMC (9D). Thegeneral format of the gauge analysis trending and trend line are asdescribed in FIG. 8B-8D. Target % CV is as described in FIG. 9A.

FIGS. 10A-10D. Detergent Comparison Study: Calibrator Stability, CHAPSDetergent Subset. (10A) The subset of the calibrators formulated withthe various CHAPS detergents (a subset of the sixteen CHAPS-formulatedcalibrators) is shown on the Variability Chart with percent stability asa function of detergent identity, day of study (time point, in days),and detergent concentration relative to CMC. Analysis, layout andspecifications are as indicated in FIG. 8A. Gauge analysis trending forpercent stability is shown for detergent identity (10B), day of study(10C), and detergent concentration relative to CMC (10D). Gauge analysisformatting is as described in FIG. 8B-1D.

FIGS. 11A-11D. Detergent Comparison Study: Student's T-test, CalibratorStability, CHAPS Detergent Subset. Individual pairwise comparisons ofmeans were computed using Student's T-tests using JMP 9.0.2 statisticalsoftware. Groups that are different from the selected group appear asthick gray circles. Groups that are not different from the selectedgroup appear as thin circles. The selected group appears as thickcircle. The four CHAPS concentrations tested are 2.83 mM, 4.76 mM(standard calibrator concentration), 8.00 mM (CHAPS CMC), and 13.44 mMare shown in (11A), (11B), (11C) and (11D), respectively, and shown inpurple typeset. The Means Comparison report for each pair of comparisonsis shown below each chart. A statistically significant difference of themean for any given comparison is p<0.05.

FIG. 12 shows a table illustrating descriptive statistics for adetergent comparison experiment

FIG. 13 shows a Material Variation Study: Overview of the ExperimentalDesign. The composition of the two collections of raw materials is shownas the “Red Team” and the “Blue Team”. The “Red Team” represents astandard grade of reagents, with the exception of the use of USP, Ph.Eur. (GMP) grade of water in formulating each raw material. The “BlueTeam” represents a test grade of reagents, with the exception of the useof the standard (HPLC) grade of water in formulating each raw material.Individually, the indicated lots/grades of each raw material from onecollection of raw materials were systematically tested in the context ofthe other collection of raw materials.

FIG. 14. Material Variation Study: Calibrator Stability, Each MaterialSubstitution Tested. The full panel of raw material substitutions usedfor formulate the test calibrators is shown on the Variability Chartwith percent stability shown as a function of each formulation conditionand day of study (time point, in days). Formulation Condition #1 is thestandard “Red Team” formulation which uses CHAPS lot #1 and BSA lot “A”and is boxed in red. Formulation Condition #35 standard “Blue Team”formulation which uses CHAPS lot #7 and BSA lot “F” and is boxed inlight blue. The survey of individual substitution of the indicated lotof CHAPS and BSA into the context of the “Red Team” standard grade ofraw materials is indicated by Conditions #4-9 and #16-20, respectively,with the appropriate comparison being Condition #1. The survey ofindividual substitutions of the indicated lot of CHAPS and BSA into thecontext of the “Blue Team” test grade of raw materials is indicated byConditions #10-15 and #21-25, respectively, with the appropriatecomparison being Condition #35. Individual substitution of indicated lotof Tris, DTT, sodium chloride, water grade, glycerol and the absence ofProClin-300 are indicated by Conditions #2-3, #26-27, #28-29, #30-31,#32-33 and #34/36, respectively. Percent stability of formulatedcalibrators is calculated relative to frozen kit calibrators. Red Teamraw material substitutions are boxed in grey, and Blue Team raw materialsubstitutions are boxed in light grey. CHAPS and BSA lots surveyed thatare not raw materials found in the Red or Blue Team collections of rawmaterials are boxed separately. Conditions in which Proclin-300 areboxed as well. Arrows show representative comparisons of raw materialsubstitution conditions to the Red and Blue team collection of rawmaterials, respectively. The provisional 97%-103% calibrator stabilityspecification is indicated as in FIG. 8A.

FIGS. 15A-15B. Material Variation Study: Calibrator Stability Trending,Part 1. Gauge analysis trending for percent stability is shown formaterial variation formulation condition (15A) and day of study (15B).The provisional 97%-103% calibrator stability specification is indicatedas in FIG. 8A.

FIGS. 16A-16C. Material Variation Study: Calibrator Stability Trending,Part 2. Gauge analysis trending for percent stability is shown for CHAPSlot number designate (16A), BSA lot number designate (16B), and theinteraction of CHAPS lot number designate and BSA lot number designate(16C). The provisional 97%-103% calibrator stability specification isindicated as in FIG. 8A.

FIG. 17. Material Variation Study: Calibrator Stability, Parsed by Lotsof CHAPS, BSA and Water Grade. The percent stability was plotted on they-axis as a function of time on the x-axis. The vertical panels showmaterial variation in the CHAPS lot usage, and the horizontal panelsshow material variation of the BSA lot usage. Material variation bywater vendor usage is shown in traces per the legend to the right.

FIGS. 18A-18F. Material Variation Study: Calibrator Stability Trending,Part 3. Gauge analysis trending for percent stability is shown for watervendor (18A), Tris vendor (18B), DTT vendor (18C), sodium chloridevendor (18D), glycerol vendor (18E), and absence/presence of ProClin-300(18F). The provisional 97%-103% calibrator stability specification isindicated as in FIG. 8A.

FIG. 19. Material Variation Study: Calibrator Stability, Parsed by Lotsof CHAPS, BSA and Glycerol Grade. The percent stability was plotted onthe y-axis as a function of time on the x-axis. The vertical panels showmaterial variation in the CHAPS lot usage, and the horizontal panelsshow material variation of the BSA lot usage. Material variation byglycerol vendor usage is shown in traces per the legend to the right.

FIG. 20. Material Variation Study: Calibrator Precision, Each MaterialSubstitution Tested. The full panel of raw material substitutions usedfor formulate the test calibrators is shown on the Variability Chartwith precision (% CV) shown as a function of each formulation conditionand day of study (time point, in days). The presentation of the resultsis as described in FIG. 14. The grand mean of the coefficient ofvariation (% CV) for all samples in this study was 1.89% (see also FIG.22A) and is shown as a dashed line. A gray hatched line shows the target% CV of 0.00%.

FIGS. 21A-21E. Material Variation Study: Calibrator Precision, Trending.Gauge analysis trending for precision (% CV) is shown for materialvariation formulation condition (21A), day of study (21B), CHAPS lotnumber designate (21C), BSA lot number designate (21D). The presentationof the results is as described in FIG. 20. The interaction of the CHAPSlot and BSA lot is shown by the effect on standard deviation of thetwelve mean % CV measurements across all timepoints (21E); see also FIG.22B. For the standard deviation plots (E), the darker lines connect thesquare root of the mean weighted variance for each effect. The ovalsindicate the spread in the standard deviations of the BSA CHAPS lotswith the BSA lot A or F, respectively. The numbers indicate formulationconditions with reciprocal effects on the standard deviations of themean % CV depending on BSA and CHAPS raw material combinations used.

FIGS. 22A and 22B are tables showing descriptive statistics for thematerial variation study described herein.

FIG. 23. Response Surface Design: The Experimental Design. A conceptualillustration of a central composite design (CCD) for three hypotheticalfactors is shown as three-dimensional cube (x, y, and z) enclosed by asphere. The imbedded factorial design with center point is shown areindicated by the vertices of a cube with a center point. The centerpoint is located in the exact center of both the cube and the sphere.The group of “star points” (also known as axial points [see inset] areindicated by “a” and “A”) reside on the surface of the sphere. The starpoints are at some distance from the center based on the propertiesdesired for the design and the number of factors in the design. The starpoints establish new extremes for the low and high settings for allfactors and are surveyed in conjunction with the midpoint concentrationsof the other effectors. For this specific, rotatable CCD design withfour effectors, the axial concentrations are twice the distance from thelow/high factorial level to the midpoint. The five effectorconcentrations for each of the four effectors in this experimentaldesign are shown in the lower right corner. The low axial, lowfactorial, center point, high factorial and high axial concentration arecoded by a, −, 0, +, and A, respectively. Thus, the midpointconcentration for all four effectors would be represented by “0000”. Theeffector concentrations circled are the standard concentrationscurrently used in the calibrator matrix (Tris, DTT, CHAPS) or thelower/upper specification limits for pH, as described in the MP-21090document.

FIG. 24 is a table showing the response surface design for effectorconcentration as described below.

FIG. 25. Response Surface Design: Calibrator Stability as a Function ofRaw Material Concentration. The percent stability the twenty sixcalibrators formulated in this study are shown as a function of CHAPSconcentration, DTT concentration, buffer pH, buffer concentration andtime (in days). Percent stability is shown as a percentage of the OD onthe indicated time point of the OD on Day 0 of the study. The nine timepoints shown were taken on Day 3, 8, 14, 30, 60, 90, 120, 150, and 180.The Day 0 time point, by definition, is set to 100%. The conditions arenumbered as in Table 3. Note that the midpoint formulation isintentionally duplicated (conditions #13/#14) as part of the designedexperiment.

FIG. 26. Response Surface Design: Absolute Value of Calibrator StabilityDifferential Relative to Target. The absolute value of the percentstability differential relative to 100% stability is shown as a functionof formulation condition number. Formulation condition number is asdescribed in FIG. 24. The gray hatched line indicates the 100% target(that is, 0% differential from target) and the red hatched lineindicates the absolute value of the provisional +/−3% specification. Thestability data points shown in blue are from the formulation conditionwith the low axial concentration (0.90 mM) of CHAPS surveyed. Thestability data points shown in blue are from the formulation conditionwith the low axial concentration (0.05 mM) of DTT surveyed. Thestability data points shown in orange are from the formulation conditionwith the low axial concentration of protons (pH 8.18).

FIGS. 27A-27E. Response Surface Design: Calibrator Stability, Trending,as a Function of Raw Material Concentration. Gauge analysis trending forpercent stability is shown for CHAPS concentration (27A), DTTconcentration (27B), buffer pH (27C), buffer concentration (27D), andtime in days (27E). The provisional 97%-103% calibrator stabilityspecification is indicated as in FIG. 8A.

FIGS. 28A-28C. Response Surface Design Study: Refined Model forCalibrator “Maximum Stability”. The analytics of the refined modeling ofpercent stability using a response surface model, including time, usingJMP 9.0.2 is shown in (28A). The parameter estimates including the pvalues are shown in (28B). Statistical significance is p<0.05. The“Prediction Profiler” was set to maximize stability for each of thetwenty-six formulations in the model in (28C). Accordingly, the“Response Limit” for “Stability” was set to “Maximize” and thePrediction Profiler was set to “Maximize Desirability”. Condition #12(the axial low CHAPS concentration) was excluded from the model at eachtime point. The effector concentrations shown are the predicted optimaleffector concentrations calculated to achieve a maximal stabilityresponse. Within each individual plot, the line within the plots (i.e.,the prediction trace) show how the predicted value changes as a functionof the value of an individual X variable. The 95% confidence intervalfor the predicted values is shown by a dotted curve surrounding theprediction trace (for continuous variables, e.g., pH). The bottom rowhas a plot for each factor, showing its desirability trace. The profileralso contains a Desirability column, which graphs desirability on ascale from 0 to 1 and has an adjustable desirability function for each yvariable. The overall desirability measure is on the left of thedesirability traces.

FIG. 29. Response Surface Design Study: Raw MaterialsConcentration-Dependent Effects on Stability. The percent stability wasplotted on the y-axis as a function of time on the x-axis. The verticalpanels show increasing CHAPS concentration from left to right, and thehorizontal panels show increasing DTT concentration from top to bottom.The five pH's tested are shown as colored traces per the legend locatedto the right.

FIGS. 30A and 30B show a table of the descriptive statistics ofcalibrator analyte values/precision for a response surface design, asdiscussed herein.

FIGS. 31A-31B. Response Surface Design Study: Calibrator Precision, as aFunction of Raw Material Concentration. The precision of the twenty-sixcalibrators formulated in this study are shown as a function of CHAPSconcentration, DTT concentration, buffer pH, buffer concentration andtime (in days). The general format of the Variability Chart format is asdescribed in FIG. 25. The grand mean of the coefficient of variation (%CV) for all samples in this study was 2.22% (See FIG. 30A) and is shownas a dashed line. A gray hatched line shows the target % CV of 0.00%.

FIGS. 32A-32H. Response Surface Design Study: Calibrator Precision,Trending, by Raw Material Concentration. The precision results weretrended by CHAPS concentration (32A), DTT concentration (32B), buffer pH(32C) and buffer concentration (32D). The grand mean % CV and target %CV are as described in FIG. 20. The mean standard deviations of the %CV's are trended for CHAPS concentration (32E), DTT concentration (32F),buffer pH (32G) and buffer concentration (32H). The analysis isanalogous to that described in FIG. 21E.

FIG. 33 is a table of a buffer/BSA survey as discussed herein.

FIG. 34 is a table showing descriptive statistics of calibratorstability at two storage temperatures as discussed herein.

FIG. 35. Buffer/BSA Survey: Calibrator Stability, Stored Frozen at −70Celsius. The stability results for the twenty-six different permutationsof the buffer composition/process and BSA survey are shown as avariability chart for the samples stored frozen at −70° Celsius. PercentStability is shown as a function of buffer composition, pre-pH process,final pH process, buffer concentration, BSA lot number and time onstability (in days). Percent stability is shown as a percentage of theOD on the indicated time point of the OD on Day 0 of the study. Theeight timepoints were taken on Days 1, 4, 7, 30, 45, 60; 90, and 120.The Day 0 time point, by definition, is set to 100%. The conditions arenumbered as in FIG. 34.

FIG. 36. Buffer/BSA Survey: Buffer/BSA Survey: Calibrator Stability,Stored at Refrigeration Temperature. The stability results for thetwenty-six different permutations of the buffer composition/process andBSA survey are shown as a variability chart for the samples stored at4-8° Celsius. Layout and analysis is as described in FIG. 35.

FIGS. 37A-37G. Buffer/BSA Survey: Calibrator Stability, Trending, StoredFrozen at −70 Celsius. The trending of the percent stability resultsusing gauge analysis for the twenty-six different permutations of thebuffer composition/process and BSA survey is shown for the samplesstored frozen at −70° Celsius. Gauge analysis is shown as a function ofcondition number (37A), time (37B), buffer composition (37C), pre-pHprocess/pH (37D), final pH (37E), buffer concentration (37F), and BSAlot number (37G). Layout is as described in FIG. 35.

FIGS. 38A-38G. Buffer/BSA Survey: Calibrator Stability, Trending, Storedat Refrigeration Temperature. The trending of the percent stabilityresults using gauge analysis for the twenty-six different permutationsof the buffer composition/process and BSA survey is shown for thesamples stored refrigerated at 4-8° Celsius. Gauge analysis is shown asa function of condition number (38A), time (38B), buffer composition(38C), pre-pH process/pH (38D), final pH (38E), buffer concentration(38F), and BSA lot number (38G). Layout is as described in FIG. 35.

FIG. 39 is a table showing descriptive statistics of OD and imprecisionat two storage temperatures, as described herein.

FIG. 40. Buffer/BSA Survey: Calibrator Precision, Stored Frozen at −70°Celsius. The precision results for the twenty-six different permutationsof the buffer composition/process and BSA survey are shown as avariability chart for the samples stored frozen at −70° Celsius.Precision is determined using all nine timepoints, including Day 0.Layout is as described in FIG. 35.

FIGS. 41A-41G. Buffer/BSA Survey: Calibrator Precision, Trending, StoredFrozen at −70 Celsius. The trending of the precision results using gaugeanalysis for the twenty-six different permutations of the buffercomposition/process and BSA survey is shown for the samples storedfrozen at −70° Celsius. Gauge analysis is shown as a function ofcondition number (41A), time (41B), buffer composition (41C), pre-pHprocess/pH (41D), final pH (41E), buffer concentration (41F), and BSAlot number (41G). Layout is as described in FIG. 35.

FIG. 42. Buffer/BSA Survey: Calibrator Precision, Stored atRefrigeration Temperature. The precision results for the twenty-sixdifferent permutations of the buffer composition/process and BSA surveyare shown as a variability chart for the samples stored refrigerated at4-8° Celsius. Analysis is as described in FIG. 40, and layout is asdescribed in FIG. 35.

FIGS. 43A-43G. Buffer/BSA Survey: Calibrator Precision, Trending, Storedat Refrigeration Temperature. The trending of the precision resultsusing gauge analysis for the twenty-six different permutations of thebuffer composition/process and BSA survey is shown for the samplesstored refrigerated at 4-8° Celsius. Gauge analysis is shown as afunction of condition number (43A), time (43B), buffer composition(43C), pre-pH process/pH (43D), final pH (43E), buffer concentration(43F), and BSA lot number (43G). Layout is as described in FIG. 35.

FIGS. 44A and 44B. Buffer/BSA Survey: Calibrator Stability/Precision,Trending, at Two Storage Temperatures. The percent stability (44A) andprecision (44B) of the set of twenty-six calibrators is compared for thefrozen (−70 Celsius) and refrigerated (+4 Celsius) storage temperatures.

FIGS. 45A and 45B. Robust Design: Transmission of Variation from Inputsto Outputs. A textbook example of Robust Design Principles is shown in(45A). The non-linear relationship between the x inputs and the youtputs is shown by the line and indicated by a green arrow and typeset.The variation of the inputs on the x-axis is indicated by red arrows andtypeset. The variation of the outputs on the y-axis is indicated by bluearrows and typeset. The non-linear relationship between the percentstability and CHAPS concentration is shown in (45B).

DETAILED DESCRIPTION

In general, described herein are compositions, kits, assays, includingrecombinant Lp-PLA2 calibrations solutions and methods of making anusing them. In particular, described herein are calibration solutionshaving a predetermined amount of recombinant Lp-PLA2 (rLp-PLA2) that isstabilized by a plurality of micelles formed of a detergent, so that therecombinant Lp-PLA2 retains activity and antigenicity (e.g., to anantibody to a native Lp-PLA2) for an extended period of time (e.g.,greater than 4 months, greater than 5 months, greater than 6 months,greater than 7 months, greater than 8 months, greater than 9 months,greater than 10 months, greater than 11 months, greater than 12 month,greater than 13 months, greater than 14 months, greater than 15 months,greater than 16 months, greater than 17 months, greater than 18 months,etc.). In some variations, described herein are calibration buffers,methods of making them, and kits and assays including them that arelow-salt calibrations buffers (e.g., having less than 1 M salt). Suchlow-salt calibration buffers may include a second detergent thatprevents aggregation of the rLp-PLA2.

The calibration solutions described herein are an advantage overexisting calibration solutions for Lp-PLA2 and particularly rLp-PLA2,which typically have a short (e.g., less than 4 months) shelf lifebefore activity of the rLp-PLA2 in the calibration solutiondeteriorates. Dilution of rLp-PLA2 in the absence of detergents resultsin irreversible gradual inactivation of the enzyme. Even in the presenceof a detergent, deterioration occurs over a comparable time scale (e.g.,between 4-6 months). The monomeric rLp-PLA2 may expose its hydrophobicinterfacial binding region or substrate binding compartment to water andcause structural collapsing of the enzyme. Further, once activity of therLp-PLA2 is lost, it cannot typically be recovered.

As described herein, certain detergents, if used to form micelles, canfully protect the enzyme from the inactivation, but cannot recover theactivity of the inactivated enzyme. Further, purified recombinantlipoprotein-associated phospholipase A2 (rLp-PLA2) expressed in HEK293cells has a propensity to form oligomers in the absence of detergents orlipids by chemical cross-linking. These observations suggest that theLp-PLA2 may form non-covalent oligomers in the absence of lipids ordetergents which serve to block access for the aqueous solvent to thehydrophobic substrate binding site and therefore prevents structuralcollapsing. Further, dilution inactivation of the enzyme can beprevented in the presence of LDL or HDL suggesting that Lp-PLA2association with lipoprotein particles (LDL and HDL) is necessary forLp-PLA2 to maintain its enzymatic activity in human plasma.

Abbreviations used herein include: BSA (bovine serum albumin); CHAPS,(3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate); CMC(critical micelle concentration); DMPC(1,2-dimyristoyl-sn-glycerol-3-phosphocholine); DTT (Dithiothreitol;EDTA, ethylenediamine tetraacetic acid); EGS (Ethylene glycolbis[succinimidyl]succinate); ELISA (Enzyme-Linked Immuno Sorbent Assay);FBS (fetal bovine serum); HDL (high density lipoprotein); HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); LDL (low densitylipoprotein); MES (4-Morpholineethanesulfonic acid); PBS (phosphatebuffered saline); PCR (polymerase chain reaction); SDS-PAGE (sodiumdodecyl sulfate-polyacrylamide gel electrophoresis); SNS (sodium1-nonane sulfonate); TBS (Tris buffered saline); TCEP(tris(2-carboxyethyl)phosphine hydrochloride); TMB (3.3′,5.5′-tetramethylbenzidine); Tris (tris(hydroxymethyl)aminomethane).

Lipoprotein-associated phospholipase A₂ (Lp-PLA₂) is a Ca²⁺ independentplasma group VII lipase (Lp-PLA₂G7) bearing the structure similaritywith other members of the phospholipase superfamily. The pathologicalroles of Lp-PLA₂ in cardiovascular diseases (CVD) are presumablyattributed to the generation of inflammatory hydrolysis products,lysophosphatidyl cholines and oxidized free fatty acids. The majority ofthe circulating human Lp-PLA₂ in blood is synthesized by macrophages andthe matured enzyme is a 45-50 kD glycosylated protein. Normally, thesecreted enzyme in the plasma has been shown to associate with highdensity lipoproteins (HDL) and low density lipoproteins (LDL) in theratio of about 1:2. Clinical studies have suggested that thepathogenicity of Lp-PLA₂ may be affected by the pattern of lipoproteinaffiliation (10) and that the ratio of Lp-PLA₂ in lipoproteins mayaffect the enzymatic activity and determine its physio-pathologicalfunctions in humans. Recent publication has been shown that thecomposition of the cell membrane or lipid vehicles affects theassociation of Lp-PLA₂ and its activity. Further, it has been reportedthat Lp-PLA₂ can migrate between lipoproteins and it has beenhypothesized that HDL may act as a transport system distributing Lp-PLA₂between LDL particles. Therefore understanding the complex interactionsbetween Lp-PLA₂ and lipids will be important in the design of diagnosticdevices and enzyme-modulating therapeutics.

The amino acid residues of Lp-PLA₂ that are involved in the interactionwith lipoproteins have been mapped out by mutagenesis and peptide amidehydrogen-deuterium exchange mass spectrometry (DXMS). Interestingly, themapped residues are shown to compose parts of the interfacial bindingregion of the enzyme identified by x-ray diffraction studies of thecrystal structure. The majority of the amino acid residues consisting ofthe interfacial binding region are very hydrophobic. It can be expectedthat the exposition of this interfacial binding region to aqueous phasewill cause high energy potential and, therefore, induce instability ofthe protein. Thus, it is likely that the enzyme must have a mechanism toprotect its hydrophobic region once released from the chaperon.recombinant Lp-PLA₂ may be expressed in HEK293 in order to study theinteraction between the enzyme and lipids or detergents.

Materials.

1-myristoyl-2-(4-nitrophenylsuccinyl)-sn-glycero-3-phosphocholine (14:0NPSPC) was purchased from Avanti Polar Lipids, Inc. (Alabaster, Ala.).The 10×300 mm Superose-6 column was manufactured by GE Healthcare LifeSciences (Piscataway, N.J.). Rabbit anti-Lp-PLA₂ polyclonal antibodieswere originally obtained from GlaxoSmithKline and also purchased fromCayman Chemicals (Ann Arbor, Mich.). Apolipoproteins were acquired fromBiodesign (Saco, Me.) or Lee BioSolutions (St. Louis, Mo.). Bothrecombinant and lipid-free human serum albumins (HSA) were obtained fromSigma-Aldrich (St. Louis, Mo.). PLAC Test and the Colorimetric ActivityMethod (CAM) assay kit for the quantitation of Lp-PLA₂ are the productsof diaDexus Inc. Recombinant Lp-PLA₂ and C-terminal His-tag Lp-PLA₂ werealso made by diaDexus Inc. as the components of PLAC test kit. Otherequipment or reagents were indicated in the text.

SDS-PAGE, Western Blotting and Protein Concentration Determination.

All SDS-PAGE were performed by using 4-12% Bis-Tris gradient gels(Invitrogen, San Diego). Gels were blotted on to nitrocellular membranesin a buffer (pH 7.5, containing 25 mM Bicine, 25 mM Bis-Tris, 1 mM EDTAand 0.05 mM chlorobutanol) for 1 hr at 50 volts. Western blots wereanalyzed by using rabbit anti-Lp-PLA₂ polyclonal antibody or asindicated in the figures. All protein concentrations were determined byusing either micro BCA or modified Bradford protein assays (PierceBiotechnology) following the manufacturer's protocols. Both assays gavesimilar results for rLp-PLA₂.

HisGrap-Enzyme-Linked ImmunoSorbent Assay (HisGrap-ELISA) and PLAC TestAssay.

For HisGrap-ELISA, chromatography fractions were loaded and incubated in96-well HisGrap nickel coated plates (Pierce Biotech, Rockford, Ill.)overnight with shaking. Plates were washed with 300 μl/well TBS, pH 7.4,containing 0.05% Tween-20 (TBS/T) for 6 times and incubated with 100 μlof primary rabbit polyclonal anti-Lp-PLA₂ antibody at 1 μg/ml each inthe same TBS/T buffer containing 3% BSA and 0.1% Proclin-300 for 3 hr atthe room temperature. The plates were then washed as described beforewith the same TBS/T buffer and further incubated with 100 μl of thesecondary antibody (goat anti-rabbit, Jackson ImmunoResearchLaboratories, West Grove, Pa.,) labeled with horseradish peroxidase(HRP) diluted at 1:15,000 in the same TBS/T/BSA buffer for 1 hr. Theplates were further washed 9 times with 300 μl/well of the same TBS/Tbuffer and incubated with 100 μl of TMB substrate for 5-20 minutes atthe room temperature in dark. The reactions were stopped with 100μl/well of 1 M HCl and concentrations were determined by reading of theplate in a SPECTRAmax M5 plate reader at 450 nm (Applied Biosystems,Foster City, Calif.).

For PLAC Test, briefly 1-40 μl (depending on the concentration) of eachsample containing rLp-PLA₂ were applied onto the assay plate wells andthe plate was incubated for 10 minutes at room temperature. Two hundredmicro liters of the anti-rLp-PLA₂ antibody-HRP conjugate solution wereadded to each well and the plate was incubated at room temperature for 3hr without sealing. The plate was then washed with TBS/T buffer for 4times and incubated with 100 μl of TMB substrate solution for 20 minutesat the room temperature in dark. The reaction was stopped by adding 100μl of 1 M HCl each well and concentrations were determined by reading ofthe plate in a SPECTRAmax M5 plate reader at 450 nm.

Enzyme Kinetic Assay and Analysis.

All of recombinant Lp-PLA₂ (rLp-PLA₂) enzyme kinetic assays in the studywere carried out by using the CAM assay kit developed by diaDexus, Inc.Basically, in a 96-well plate, reactions were started by adding 110-134μl of the reaction buffer to each well containing 1-25 μl of Lp-PLA₂samples according to the protocol by the manufacturer. The volumes ofenzyme and reaction buffer were depended on the individual experiment.The reactions were followed at OD405 nm (absorbance) in a SPECTRAmax M5plate reader and the steady state reaction rates of the first 3 or 5minutes depending on the experiments were averaged. The data wereprocessed and analyzed by using Microsoft Excel and GraphPad Prism(version 4).

Chemical Cross-Linking of rLp-PLA₂.

Purified rLp-PLA2 in 50 mM Tris, pH 8.0, with and without 10 mM CHAPSwas diluted 1340 or 1460 fold to the final concentration of 1.0 μg/ml in50 mM sodium phosphate with and without 10 mM CHAPS or 1% Tween-20, pH7.6, containing 100 mM sodium chloride and 3 mM EGS. The mixtures wereincubated at room temperature for 45 min and ethanol amine was added tothe final concentration of 0.5 M to stop the reactions. The mixtureswere then concentrated about 10 fold through a 20-kD cutoff iCONconcentrator. Thirty μl of each sample were mixed with 10 of 4-foldSDS-PAGE loading buffer containing 200 mM DTT and 20 mM TCEP andincubated at 60° C. for 15 minutes and subjected to electrophoresis.

FPLC Fractionation.

Fractionation chromatography was carried out on an Akta10 or Akta100 byusing a 10 mm×300 mm Superose-6 column at room temperature with the flowrate of 0.3 ml per minute. Three different buffer systems (A: 50 mMsodium phosphate, pH 7.4, containing 100 mM NaCl, 2 mM EDTA and 0.01%sodium azide; B: PBS, pH 7.2; C: 50 mM Tris/HCl, pH 8.0) were used andno significant difference was observed. Fifty to two hundred μl ofsamples were injected per run depending on the Lp-PLA2 concentrations ofthe samples after the column was equilibrated with the running buffer.Fraction collection was started at 21 minutes (the column void volume)after the sample injection and the collection volume was 0.6 ml/tube.

Preparation of LDL and HDL Lipoproteins Devoid of Lp-PLA2 EnzymaticActivity.

Concentrated human LDL and HDL were purchased from Lee BioSolutions inSt. Louis. According to the manufacturer, LDL and HDL were prepared fromfresh human plasma by undisclosed precipitation methods. Both the LDLand HDL showed one major band by Helena lipoprotein cellulose acetateelectrophoresis. Characterization indicated thattriglyceride/cholesterol ratios were 0.86 and 0.40 for LDL and HDLrespectively. The lipoproteins were stored at −40° C. and shipped on dryice. The purchased lipoproteins were thawed and subjected toinactivation by incubation with 20 mM Pefabloc SC (Roche AppliedScience, Indianapolis) in PBS, pH 7.2, at 4° C. overnight. The PefablocSC inactivated lipoproteins were then dialyzed extensively with a 10 kDcutoff membrane in 1000 fold volume excess of buffer containing 50 mMphosphate, pH 7.2, and 150 mM sodium chloride with 3 exchanges at 4° C.The inactivated lipoproteins were found to have less than 10% of theoriginal endogenous Lp-PLA2 activity by the CAM assay. Both lipoproteinswere further diluted to the desired concentrations before used in eachexperiment.

Results

Association of Lp-PLA₂ with Detergent Micelles.

To estimate the molecular size of the rLp-PLA₂ expressed in HEK293cells, the purified enzyme was subjected to fractionation by a 10×300 mmSuperose-6 column in the presence and absence of 10 mM CHAPS. Theresults indicated that the same enzyme was eluted very differently underthe various conditions (FIG. 1A). According to the molecular weightreference, rLp-PLA₂ was eluted between the chicken ovalbumin (44 kD) andhorse myoglobin (17 kD) in the presence of 10 mM CHAPS and between thebovine thyroglobulin (670 kD) and bovine Ig-globulin (158 kD) in absenceof the detergent (chromatography of molecular markers not shown). Theexpected molecular weight of Lp-PLA₂, not including the glycosylationoligosaccharide chains, is about 48 kD. To further understand theretention time shift, we resolved the enzyme by the same procedure withdifferent detergents. The results showed that the column retainedrLp-PLA2 differently with different detergents (FIG. 1). Detergents withlarger micelle molecular weight eluted rLp-PLA2 earlier from the column.This indicates the association of rLp-PLA2 with the micelles of thedetergents. However, the molecular size of the rLp-PLA2 in the absenceof the detergents seems larger than that of the complex of enzyme anddetergent micelles tested. This suggests a possibility that the enzymemay form oligomeric structures or aggregate in the absence ofdetergents. We also fractionated the unpurified rLp-PLA2 from the cellcultural supernatant of HEK293 and it gave the same results as thepurified enzyme under the same conditions (results not shown). Anotherobservation was that the recovery yield based on the CAM enzymatic assaywas much lower when rLp-PLA2 was fractionated in the absence ofdetergents (FIG. 1). In the absence of detergents, only about 23% ofrLp-PLA2 activities were recovered compared to 60-146% recovery in thepresence of detergents. To investigate the lost rLp-PLA2 in the absenceof detergents, purified rLp-PLA2 with a His-tag at the C-terminal wassubjected to fractionation and the fractions were assayed by both theCAM assay and the His-ELISA using rabbit anti-Lp-PLA2 polyclonalantibody. When rLp-PLA2 was fractionated in the absence of detergents,the results indicated that two mass peaks (fraction 16-18 and 21-23)were shown by the His-ELISA but only one activity peak (fraction 16-18)was seen by the CAM assay (FIG. 1B). That is, the lower molecular weightmass peak (fraction 21-23) contained no enzymatic activity. However,when the enzyme was fractionated in the presence of 10 mM CHAPS in thesame buffer, no mass or enzymatic activity at fraction 16-18 was seenbut both mass and enzymatic activity were detected at the fraction 21-23(FIG. 1C). This suggests that the lower molecular weight peak (fraction21-23), which probably comes from the higher molecular weight peak(fraction 16-18), losses its activity irreversibly in the absence ofdetergents. In the presence of detergents, rLp-PLA2 is probablydeoligomerized and stabilized by the formation of the complex withdetergent micelles.

Inactivation of rLp-PLA2 by Dilution in the Absence of Detergents.

It was found that freshly prepared rLp-PLA2 stored in the presence orabsence of detergents had no difference in specific activity whenassayed with CAM (results not shown). However, the enzyme stored in theabsence of detergents at 4° C. lost its activity faster, especially whenthe concentration was low (results not shown). To further investigatethe decrease of rLp-PLA2 specific activity in the absence of detergents,the enzyme was subjected to dilution to the final concentration between1-3 μg/ml in PBS, pH 7.2, and the changes of the enzymatic activity andimmuno-reactive mass were followed. The immuno-reactive mass of Lp-PLA2was quantified by using the PLAC kits that only recognized thenon-denatured form of the enzyme (conformational). FIG. 3 shows that theenzyme gradually lost its activity and immuno-reactive mass in twophases. Upon dilution, the enzymatic activity and the immuno-reactivemass had a sharp decline phase (about 1-2 days of incubation at 4° C.)and then the inactivation rate decreased and transferred to a slowerphase (FIG. 2). The final normalized losses in both activity andimmuno-reactive mass were in the range of 50-75% at the fifteenth day ofincubation. Actually, for each reaction, the inactivation rates andfinal losses of the enzymatic activity and immuno-reactive mass variedwith different experimental conditions depending on the final dilutedenzyme concentration (see the following experiments), the storageconditions of the enzyme, the dilution buffer components and incubationtemperature, etc.

The Effects of Detergents on the Activity of rLp-PLA2.

The effects of detergents on the dilution inactivation of rLp-PLA2 wereinvestigated. When 10 mM CHAPS was included in the dilution buffer, noinactivation was observed for the diluted rLp-PLA2 at 1 μg/ml (FIG. 3A).However, the addition of 10 mM CHAPS into the inactivated enzymes onlyrecovered a very small portion of the lost activity but it did preventthe enzyme from further inactivation during the extended incubation(FIG. 3A). In addition to CHAPS, several other non-ionic detergents,such as Tween-20, Triton X-100 and digitonin, were also found protectivein the dilution inactivation of rLp-PLA2 (data not shown). Detergentswith high CMC were less effective than those with lower CMC. In anexperiment of dilution inactivation for rLp-PLA2, the diluted enzyme wasincubated in buffers containing variable detergent concentrations from0.15 mM to 10 mM. The rate of enzyme inactivation was found to beconcentration dependent for CHAPS (CMC=6 mM) and deoxycholate (CMC=1.5mM) but not for Triton X-100 (CMC=0.3 mM), Digitonin (CMC=0.09 mM) andTween-20 (CMC=0.06 mM) (FIG. 3B). This suggests that detergent micelles,instead of monomeric detergent, are the stabilizer of rLp-PLA2 molecule.

The Effects of the Protein Concentration on the Activity of rLp-PLA2.

At high concentrations (>0.5 mg/ml), rLp-PLA2 is fairly stable even inthe absence of detergents (observation not shown). In the dilutioninactivation of the recombinant Lp-PLA2, the inactivation rates aredependent on the final diluted concentration of the enzyme. Theconcentration effect on the rLp-PLA2 dilution inactivation isillustrated in FIG. 4A. The rate and final loss of the rLp-PLA2inactivation upon dilution varied in the enzyme concentration range of0.6-5 μg/ml. The inactivation rates became relatively independent offinal enzyme concentrations at both ends of the above concentrationrange. This can be better demonstrated by plotting the residual residuepercentage of the rLp-PLA2 activity after the enzyme was diluted andincubated at 4° C. for ten days against the protein concentrations (FIG.4B). In the logistic scale of concentration, it can be fitted into asigmoidal curve. There is a sensitive range between 1 and 5 μg/ml. Thesaturation at both concentration ends may indicate that there is adynamic equilibrium between the stable and unstable forms of rLp-PLA2,which shifts depending on the concentration of the enzyme. Since theinactivation is due to structural disruption by solvent andirreversible, it should be a reaction of first order kinetics, that is,concentration independent. When the enzyme concentration decreases to acertain level, the equilibrium is shifted to the unstable form and thenthe irreversible inactivation rate becomes concentration independent.When the concentration of rLp-PLA2 increases, the rate of inactivationis reduced due to the equilibrium shifting to the stable form of theenzyme. Most likely, the stable and unstable forms of Lp-PLA2 shouldrepresent the oligomerized and the dissociated enzyme respectively sincethe dilution usually causes dissociation and vice versa.

Protection of rLp-PLA2 Activity by Lipoproteins.

Lp-PLA2 protein has been shown to associate with LDL and HDL in humanplasma (9). Experiments were designed to reveal if LDL and HDL wouldprevent rLp-PLA2 from the inactivation during the dilution intonon-detergent containing buffers. Purified rLp-PLA2 was diluted in 50 mMsodium phosphate buffer, pH 7.2, containing 150 mM sodium chloride and 2mM EDTA at the final concentration of 0.5 μg/ml enzyme and incubated at4° C. for 2 days. The experiments were carried out in the presence ofvarious concentrations of fractionated LDL and HDL (devoid of endogenousLp-PLA2 activity). It was indeed found that the dilution inactivation ofrLp-PLA2 could be averted in the presence of either LDL or HDLparticles. FIG. 5 shows that human LDL or HDL at concentrations as lowas 1.4 and 0.14 mg/dL of triglyceride respectively fully protected therLp-PLA2 activity during the dilution in the phosphate buffer. Nosignificant activity losses were observed after the two day period ofincubation at 4° C. in the LDL or HDL containing buffer while more than90% of the original activity vanished in the control buffer. However,unexpectedly higher concentrations of LDL or HDL reduced the protectioncapability possibly due to the proteolysis of the recombinant enzyme(data not shown).

The Effects of Chaotropic Agents on the Activity of rLp-PLA2.

According to the gel permeation experiments, detergents could reduce themolecular weight of rLp-PLA2 and stabilize its activity. To investigatethe connection between the deoligomerization and stabilization effectsof detergents, rLp-PLA2 was diluted and incubated at 4° C. in thepresence of 1 M sodium salts of fluoride, bromide, chloride, iodide,nitrate, sulfate (0.5 M) and thiocyanate. While detergents were found tostabilize rLp-PLA2, anions destabilizing protein-protein interactions,such as SCN⁻¹ or I⁻¹, were found to promote the inactivation of theenzyme. The inactivation of the diluted rLp-PLA2 during the incubationat 4° C. was significantly accelerated by including 1 M of NaSCN or Nalin the incubation buffer (FIG. 6). This is not due to the added sodiumsalt concentration because no other salts had effects on the stabilityof the enzyme. None of the above chemicals (up to 1 M) was foundinhibitory to the enzymatic activity of rLp-PLA2 either (results notshown). The experiment suggests that the protein-protein interactionbreaker such as SCN⁻¹ or I⁻¹ actually destabilizes rLp-PLA2. It can beinferred that rLp-PLA2 tenders to form a dimer or oligomers during theincubation but, if the self-interaction is prevented or interrupted bychaotropic agents, the monomeric enzyme will be denatured, possibly dueto exposure of the hydrophobic substrate binding site to aqueoussolvents.

Chemical Cross-Linking of rLp-PLA2.

To further confirm the formation of the oligomeric rLp-PLA₂ duringdilution, the highly purified enzyme was diluted into buffers containinga chemical cross-linker, ethylene glycol bis[succinimidylsuccinate](EGS), with and without detergents. FIG. 7 shows the results of thecross-linking experiment. First of all, when rLp-PLA2 was diluted to thefinal concentration of 1 μg/ml in the absence of detergents, onlyoligomers with molecular weight >98 kD were detected on the Western Blotby rabbit anti-Lp-PLA2 antibody. No monomeric (48 kD) and only a lowamount of dimeric (98 kD) rLp-PLA2 were seen. Second, the extent ofrLp-PLA2 oligomerization observed was different when stored at differentconditions. Enzyme stored in buffer containing 5 mM CHAPS had a loweroligomerized molecular weight than enzyme stored in the detergent-freecondition although both were diluted into the same cross-linking bufferat the same final concentration. Third, in the presence of 10 mM CHAPS(or 1% Tween-20, data not shown), the majority of rLp-PLA2 stayedmonomeric after cross-linked by EGS. Again, the enzyme stored in thepresence of 5 mM CHAPS was almost free of oligomeric bands whencross-linked in buffer containing detergents while the detergent-freeenzyme still had significant amounts of high molecular weight specieswhen cross-linked in the same buffer. These results prove that rLp-PLA2does quickly self-associate and form polymers upon dilution in theabsence of lipid substrates or detergents. The detergents do not reducethe reactivity of EGS in the cross-linking of rLp-PLA2 because thecontrol experiments to internally cross-link IgG by EGS were not alteredby the presence of the same detergents (data not shown).

In the characterization of rLp-PLA2 by size exclusion chromatography, itwas found that the recovery yield in the absence of detergents was verylow as shown by the CAM activity of the collected fractions (FIG. 1). Byincluding 10 mM CHAPS in the chromatography buffers, not only therecovery yield was improved but also the molecular size of the rLp-PLA2was reduced. This suggests that the enzyme may not exist as themonomeric form in the absence of detergents. Indeed, fractionation ofthe C-terminal His-tag rLp-PLA2 in the absence of detergents andassaying the fractions by HisGrap-ELISA using rabbit anti-Lp-PLA2polyclonal antibodies, which detects both the native and denaturedrLp-PLA2, we demonstrate that a lower molecular weight mass peak withoutenzymatic activity was missed by the CAM assay (FIGS. 1A-1C). It isunlikely that the mass without activity comes from the impurity thatcross reacts with the polyclonal antibodies because the recombinantprotein has been purified to highly homogeneous purity and subjected toSDS-PAGE and Western Blotting analyses (data not shown). The resultssuggest that it is the monomeric rLp-PLA2 that may not be stable in theabsence of detergent and it can also be inferred that the enzyme mayform oligomers in the absence of detergents in order to keep thehydrophobic sites away from aqueous solvent. When diluted, there will bemore monomeric rLp-PLA2 formed because of the increase in the rate fordissociation and the decrease in the rate for oligomer formation. Thus,dilution will cause more inactivation of the enzyme in the absence ofdetergents. This is indeed the case observed in our study. To explainthe dependence of the inactivation rate on the rLp-PLA₂ concentrations,one model is that oligomization and dissociation are reversible stepsbut the denaturation is an irreversible step. At high proteinconcentration, the rate of oligomer formation is fast and the monomericrLp-PLA2 is less abundant and, therefore, the enzyme is stable. Dilutionor breaking protein-protein interaction will increase monomeric rLp-PLA2and, in the absence of substrate or detergents, it will result in theinactivation of the enzyme. It is possible that the monomeric rLp-PLA2has widely open hydrophobic regions, such as the interfacial orsubstrate-binding site, as illustrated by the crystal structure. Accessof these hydrophobic regions by aqueous solvents would result in thedisruption of the rLp-PLA2 molecular structure and, thus, the enzymewould block these hydrophobic regions by self-association oroligomerization to keep the water molecules away in the absence of otherhydrophobic entities. In the presence of other hydrophobic particlessuch as detergent or substrate micelles, or lipid particles, therLp-PLA2 molecule would form complexes with these compounds to cover itshydrophobic regions. Unlike enzymes such as Rhizomucor miehei lipase,which has a “lid” to cover its active site in the absence of substrate,Lp-PLA2 probably has to form complex to shield aqueous solvent from theempty hydrophobic substrate binding site. If a proper complex partner isnot available, the monomeric Lp-PLA2 will form a self-complex and itdoes not stop as a dimer but can extend to oligomers with different unitlength. By associating together, Lp-PLA2 molecules reduce thehydrophobic surface area exposed to water and minimize the disruptiveeffect. The deuteration experiments by using DXMS method have shown thatthe active site residues of Lp-PLA2 barely exchange with solvent. Thisdoes indicate that the active site of Lp-PLA2 is in a closed form.Self-oligomization was previously reported for a Group VICa²⁺-independent cytosolic phospholipase A2 although the functionalbenefit for the enzyme was not discussed. Self-oligomerization is notuncommon for biological active proteins. One of the best studiedexamples is insulin. In most of the cases, protein self-associationplays important roles in protein biosynthesis and preservation ofprotein functional activities. In other cases, protein self-associationis to form specific structures such as apolipoprotein A (ApoA). Insummary, the results shown here demonstrate that if Lp-PLA2 monomersfail to form a complex or oligomer during dilution into lowconcentration, in the absence of lipid substrates or detergents, theenzyme will go through irreversible denaturation possibly initiated bythe disruption of hydrophobic regions in the aqueous milieu. In thepresence of detergents, Lp-PLA2 will associate with detergent micellesand stabilize as a monomer. The roles of lipid particles such as LDL orHDL in human plasma may be just like detergent micelles in theseexperiments; the lipid particles act as the chaperones to stabilize theLp-PLA2 in circulation possibly by binding to its hydrophobic interface.Reducing lipids by statins or fibrates was found also reduced Lp-PLA₂mass and activity.

In addition to finding that recombinant Lp-PLA2 may be effectivelystabilized by include micelles in the buffer solution to protect therLp-PLA2, additional modifications to stabilize a calibration solutionof rLp-PLA2, including salt content, additional detergent, pH, and thelike have been examined to determine how to prepare stable calibrationsolutions with a long shelf-life.

A series of four real-time stability studies were performed to identifystability factors for an Lp-PLA2 assay, and particularly the recombinantLp-PLA2 calibrators used for the assays. In many of these examples, theLp-PLA2 assays are mass (e.g., immune-) assays. In these example, theLp-PLA2 assay may be referred to as a “PLAC ELISA kit” and theassociated calibration standard(s).

In the examples described below, a combination of component swappingassays and designed experiments were utilized to characterize rawmaterials and their possible (e.g., desired or optimal) concentrations.One goal was to utilize robust design principles to maximize stabilityand minimize variation of the calibrator formulation in order to provideexcellent product performance and desired expiration dating of a kit. Itshould be understood that while these studies provide actualexperimental results, other factors may also be taken into account indesigning a calibration standard. For example, other factors mayadditionally affect the stability or other parameters of interest in acalibration standard (e.g. color, turbidity, viscosity, etc.) and acalibration standard may be contemplated that takes, on the whole,multiple factors into consideration. An individual component may (or maynot) be used under its optimal performance. The result may be that acomponent or formulation described herein may be useful at a differentconcentration or in combination with other factors than what the data,on face value, may suggest. Additionally, any parameter or componentreferred to or described herein is recognized as one that may becontemplated for generating a calibrator formulation regardless of aspecific experimental result. In particular, as some unpredictabilityexists with any experimental system (no matter how well designed orexecuted), an individual experimental result should not be taken as theonly possible outcome.

Thus, the calibrator solutions and assays and kits including them arenot limited to use with one particular type of assay (e.g., a “PLAC testELISA assay). Other assays have been examined for calibrator performanceas described herein, including a “Auto-CAM” enzymatic activity assay forLp-PLA2. Thus, the same calibrators may be used in other platforms forthe analyte Lp-PLA2 involving clinical analyzers, and these calibratorresults may be applicable to the other platforms; calibrators may becorrected for differences in assay temperatures, ionic strengths ofreagent systems, identities of the detergents used (and theircorresponding critical micelle concentrations), different length assaytimes and intrinsic on-rates/off-rates for a given antibody:antigenequilibrium binding state (or, more likely, its non-equilibrium bindingstate) at any given set of assay conditions, etc.

The first stability study (Example 1, below) described is a systematiccomparison of a panel of detergents in the context of an existingcalibrator diluent formulation. The effect on calibrator stability ofsubstituting a battery of detergents into an existing calibratorformulation at different concentrations was assessed. This studyincluded substituting various CHAPS analogues as well as CHAPS suppliedfrom various vendors/grades/lot numbers for the primary detergent intoan existing calibrator formulation. The results of this study identifiedperformance differences between CHAPS lots as well asconcentration-dependent effects of CHAPS on calibrator stability. Moregenerally, the results strongly suggest that detergent micelle formationmay be an important factor for Lp-PLA2 protein stability in someformulations.

The second stability study (Example 2, below) was designed to explorethe effect of calibrator diluent raw material quality on calibratorlong-term stability. Thirty-six separate raw material combinationssourced from at least two different vendors or grades were compared. Inthis study, the raw materials tested included CHAPS, BSA, DTT, sodiumchloride, water, glycerol and ProClin-300 (present/absent). The resultsconfirmed that the CHAPS detergent is an important factor for calibratorstability in some cases. In addition, different combinations of CHAPSand BSA interacted synergistically to affect both stability andprecision. This study also provided evidence that choice of glycerol(including grade of glycerol) can affect calibrator stability.

The third stability study (Example 3, below) was a response surfacedesign experiment that explored the effects on calibrator long-termstability of experimentally manipulating Tris buffer pH, Tris bufferconcentration, CHAPS concentration and DTT concentration. First, theexperimental results indicated that incrementally increasing the CHAPSconcentration above a standard concentration has a positive effect oncalibrator stability. Conversely, decreasing the CHAPS concentrationbelow a standard concentration has an adverse effect on calibratorstability. Second, the labile reducing agent DTT was identified as auseful effector of calibrator stability in some cases.

The fourth stability study (Example 4, below) explored the effects ofdifferences in Tris buffer composition, Tris buffer pH and differentgrades and/or lots of Probumin BSA. In the context of the calibratorformulation, minor perturbations in buffer composition seemingly had nodiscernable effect on calibrator stability. The effects of surveyingdifferent grades of BSA on calibrator stability suggested idiosyncraticdifferences that vary by the lot number of BSA used rather than anysystematic differences based on the grade of BSA used. Interestingly, aprocess change in the starting and final pH of the calibrator matrixconferred enhanced precision in this study.

Taken together, the results of these studies suggest that raw materialsin calibrator diluent may include CHAPS detergent at appropriate levels,a reducing agent (DTT), and glycerol. A combination of additionalincoming quality control specifications/testing (CHAPS purity, glycerolquality), manufacturing process controls (ensuring DTT integrity) andadditional critical raw material validations studies (increased CHAPSconcentration, pH adjustment) may be useful. In these studies, thepercent stability and precision of the calibrator formulations were usedas a more direct response rather than the indirect response of serumpercent stability. In addition, the precision of the calibrators is muchbetter than the precision of serum samples, particularly those serumswith high Lp-PLA2 analyte levels. Utilizing the stability and precisionof the calibrator formulations as direct responses should allow bothmore sensitivity in the measurement of the responses as well as ageneral reduction in the signal:noise ratio compared to assaying theserum samples as a response.

In the examples below, the following terms may be used and understood asfollows:

Coefficient of Variation (% CV) may refer to a measure of the relativevariation of distribution independent of the units of measurement; thestandard deviation divided by the mean, expressed as a percentage.

Critical Micelle Concentration (CMC) may refer to the concentration ofsurfactants above which micelles form and almost all additionalsurfactants added to the system go to micelles

Designed Experiment (DOE) may refer to experimental methods used toquantify measurements of factors and interactions between factorsstatistically through observance of forced changes made methodically asdirected by mathematically systematic tables.

Full Factorial Design may refer to A DOE that measures the response ofevery possible combination of factors. These responses are analyzed toprovide information about every main effect and every interactioneffect. The approach used in screening experiment to identify maineffectors and to identify first-degree polynomial effects.

Gauge Analysis may refer to attribute gauge analysis that gives measuresof agreement across responses in graphs summarized by one or more Xgrouping variables.

Response Limit may refer to the specification of one of the possiblegoals for a DOE response variable, such as percent stability. JMP allowsone to choose from the following goals: Maximize, Match Target,Minimize, or None.

Response Surface Design may refer to a type of DOE experiment thatallows the interactions between factors to be mapped and it identifiesquadratic (second degree polynomial) effects. It is typically used tooptimize a process and/or make it more robust.

Robust design may refer to the practice of making the response of asystem insensitive (or robust) to uncontrollable variation bydesensitizing the product to these potential sources of variation.

Variability Chart may refer to a variability chart that shows how ameasurement varies across categories. The mean, range, and standarddeviation of the data can be analyzed in each category. The analysisoptions assume that the primary interest is how the mean and variancechange across the categories.

Variance Inflation Factor (VIF) may refer to a large VIF value indicatesthat the X variable is highly correlated with any number of other Xvariables. VIF values between 1-3 are no problem; VIF's between 4-7, areproblematic and should be removed; VIF's >8, must be removed.

Example 1 Detergent Comparison Study

Purpose: To compare the effect of substituting various alternativedetergents, different CHAPS analogues and CHAPS raw materials sourcedfrom different vendors/grades in a short-term stability study.

Materials:

PLAC ELISA kit, P/N 90123, L/N 1001003

Antigen: P/N 26203, L/N 1010057

BSA: Roche Diagnostics, P/N 03117405001, L/N 70189921

Glycerol: EMD, P/N GX0185-5, L/N 41116133

Detergent Screening Kit (all Dojindo), P/N DS06, L/N CT717: CHAPS (CAS#75621-03-3), L/N CM607; n-Dodecyl-β-D-maltoside (CAS #69227-93-6);n-Octyl-β-D-glucoside (CAS #29836-26-8); Sodium cholate, monohydrate(CAS #73163-53-8); MEGA-8 (CAS #85316-98-9)

Modified CHAPS analogues (all Dojindo): BIGCHAP (P/N D043, L/N CT710;CAS #86303-22-2); CHAPSO (P/N C020, L/N CT711; CAS #82473-24-3);deoxy-BIGCHAP (P/N D045, L/N CT712; CAS #86303-23-3);

CHAPS from standard vendor (all Sigma): CHAPS: P/N C3023, L/N 018K53003(lot #1); CHAPS: P/N C3023, L/N 040M5319V (lot #2); CHAPS, BioXtra: P/NC5070, L/N 18K530041V (lot #3)

Experimental Procedures:

Experimental Plan: The eleven different detergent variants in this studyincluded each of those included in Dojindo “First Choice” detergentscreening kit (CHAPS, n-Dodecyl-β-D-maltoside, n-Octyl-β-D-glucoside,sodium cholate and MEGA-8), various CHAPS analogues (Dojindo detergentsCHAPSO, BIGCHAP, deoxy-BIGCHAP), and various grades/lot numbers of SigmaCHAPS (including two lots from a current grade of CHAPS). All thedetergents were substituted into a standard calibrator diluentformulation at four concentrations each in a linear titration series.The concentration range surveyed for each detergent was based on eachindividual detergent's published critical micelle concentration (CMC).Most detergents were also tested at one concentration above the CMC andtwo concentrations below the CMC, with the single exception being theMEGA-8 detergent. The MEGA-8 detergent presented a technical challengewith respect to testing above its published CMC (58 mM). With thisconsideration in mind, the highest concentration of MEGA-8 detergentsurveyed was 50 mM. In total, forty-four different calibrator diluentformulations spiked with antigen at a single analyte concentration weretested in a short-term refrigerated stability study.

Experimental Details: The reactions were systematically assembled as twomaster-mixes to facilitate the highest detergent concentrations goinginto solution quickly into the standard calibration diluent formulation.This is due to the formulation's high ionic strength, contributedprincipally by the 2.857 M NaCl. Mastermix A (5×) was a pre-formulated,buffered isotonic salt solution into which BSA and Proclin-300 dissolveduntil a homogeneous solution is achieved. Master-mix B (1.33×) was apre-formulated, buffered high salt solution with the reducing agent DTTdissolved until a homogeneous solution is achieved. An appropriatevolume of each of the two master-mixes is added to each of theforty-four formulations with an appropriate volume of a concentrateddetergent stock solution and HPLC-grade water (if necessary) to achievethe desired final detergent concentrations for the forty-four variantsof the calibrator diluent formulation as indicated in the legend forFIG. 8A-8D. An appropriate amount of 100% glycerol is added so that thefinal concentration of all non-detergent raw materials is at theirstandard calibrator diluent concentrations per MP-21090. After mixing,the recombinant protein Lp-PLA2 was added to each formulation in orderto achieve an intended final analyte concentration of approximately 250ng/mL. The calibrator formulations were put on stability at refrigeratedtemperature. Three stability timepoints are taken at Day 0, Day 14 andDay 30 using a PLAC ELISA kit.

Results

A. Detergent Comparison Study, One Month

Eight different detergents were compared in this one month study,including four individual lots of CHAPS sourced from two differentvendors, for a total of eleven variations (FIGS. 8A-8D). Eachdetergent/vendor/grade/lot variation was assayed for calibratorstability at four concentrations, with most of the detergents beingtested using concentrations that bracket their published CriticalMicelle Concentration (CMC). The one exception was the MEGA-8 detergentdue to practical considerations of its extremely high CMC value (Seelegend to FIGS. 8A-8D). In addition to assaying at CMC, twoconcentrations lower than the published CMC and one concentration higherthan the published CMC were assayed. A linear concentration titrationwith a dilution factor of 1.68-fold was tested for each detergent inorder to specifically accommodate the fold-concentration differencebetween the standard CHAPS concentration used in the calibrator diluentformulation (e.g., 4.76 mM) and its published CMC (e.g., 8.00 mM).Published studies indicate that micelle formation by CHAPS isconcentration-dependent High salt conditions, such as those found in thecalibrator diluent formulation, favor micelle formation. In contrast,lowering the temperature, a situation that occurs upon initiating areal-time stability study, disfavors micelle formation. In thisshort-term refrigerated stability study, timepoints were taken at both14 and 30 days and then compared on a percentage basis to the Lp-PLA2analyte value of the initial time point.

A comparison of the percent recovery of the each of the forty-fourcalibrator formulations at the Day 14 and Day 30 timepoints is shown inFIG. 8A. Percent stability was calculated based on analyte valuesestablished using refrigerated finished good calibrators. Theprovisional 97%-103% calibrator stability specification is shown by thered hatched lines with the target stability of 100% relative to Day 0shown as a green dashed line (FIG. 8A-8D). The mean analyteconcentration in the CHAPS-formulated calibrators including all lots andall three timepoints was 418 ng/mL. The CHAPS (from Dojindo) and sodiumcholate were among the best performing detergents with regard toshort-term refrigerated stability when both subsequent timepoints arecompared to their individual Day 0 analyte value (FIG. 8A). Across thefour concentrations and two timepoints surveyed, the gauge analysisindicates that the mean percent stability for these two detergentstested even meets the provisional 97%-103% stability specification forpercent recovery at individual timepoints (FIG. 8B). The detergent,n-dodecyl-β-D-maltoside, was the worst performer of the group testedwith even the highest detergent concentrations not resulting in goodLp-PLA2 stability relative to Day 0 (FIG. 8A). On average, the analytevalues of the forty-four calibrators trended incrementally higher on Day30 than on Day 14 (FIG. 8C). When the percent stability data is trendedas function of detergent concentration, the detergents' collectivelyshow an interesting profile: In general, the percent stability drops offquickly once the detergent concentration drops below its individual CMC(FIG. 8D), strongly suggesting micelle formation may be necessary foroptimal Lp-PLA2 stability.

A comparison of the imprecision (e.g., by coefficient of variation [%CV], n=2 replicates per formulation condition per time point) of theeach of the forty-four calibrator formulations at each of the threetimepoints is shown in FIG. 9A. The overall mean % CV for the forty-fourdetergent conditions across all timepoints was 1.81%. Of the elevenformulation surveyed, the gauge analysis indicates that the CHAPSOdetergent demonstrates the best precision whereas the MEGA-8demonstrated the worst precision (FIG. 9B). Of the four CHAPS lotstested from two vendors, small differences in imprecision can bedetected. The Sigma lot #3 (BioXtra grade; P/N C5070, L/N 18K530041V)demonstrated the best precision, and the single lot of Dojindo CHAPSdemonstrated the second-best precision (FIG. 9C). The other two lots ofSigma CHAPS (P/N C3023; lot #1, L/N 018K53003; lot #2, L/N 040M5319V)were a manufacturing grade used for calibrator diluent production andboth showed worse mean precision than the grand mean of 1.82% (i.e., forall detergents tested; FIG. 9B). Across all the detergents surveyed inthis study, there was no obvious relationship between detergentconcentration and precision (FIG. 9C). There was a discernable trendover the forty-four formulations of the precision getting graduallyworse (i.e., higher % CV's) over the course this short-term stabilitystudy of only thirty days (FIG. 9D).

The percent stability data for the sixteen CHAPS-based calibratorformulations (four lots at four concentrations each) were parsed outfrom the stability data from other detergents and analyzed in moredetail in FIGS. 10A-10D and FIGS. 11A-11D. In general, the lot of CHAPSfrom Dojindo (P/N C008, L/N CM607) showed superior stability compared toall three lots of Sigma CHAPS at both the Day 14 and Day 30 timepoints(FIG. 10A). The worst performer was the Sigma lot #3 (BioXtra; P/NC5070, L/N 18K530041V). Across the four concentrations and twotimepoints surveyed, the gauge analysis indicates an overall trend thatthe lot of CHAPS from Dojindo showed superior stability compared to allthree lots of Sigma CHAPS tested in this study (FIG. 10A). Similar tothe analysis with the entire battery of detergent formulations (FIG.10C), the percent recovery of the sixteen CHAPS-formulated calibratorstrended incrementally higher on Day 30 than on Day 14 (FIG. 10C),possibly reflecting some effect related to day-to-day variability. Whenthe percent stability data for the CHAPS-formulated calibrators alone istrended as a function of detergent concentration, the trending indicates˜10% drop in percent stability when the CHAPS concentration drops belowa concentration (i.e., 4.76 mM, indicated in FIG. 10D as the coefficient0.595 [of CHAPS CMC value]). Above the standard concentration of CHAPS,the percent stability of the calibrators plateaus (FIG. 10D), at leastin the context of this short-term stability study of 30 days duration.

A Student's t-test was performed to compare the mean percent stabilityof the two timepoints (i.e., the mean of the percent stability Day 14and Day 30 timepoints at each CHAPS concentration) for statisticallysignificant differences (p value <0.0500; FIG. 11A-11D). The Student'st-test was the comparison selected because the primary goal was todetect if there is a difference in the mean value for each of theindividual Sigma lots compared to the single lot from Dojindo. Asecondary goal was to the Student's t-test compare the means betweenindividual lots of CHAPS from Sigma. The Dojindo lot of CHAPS showedstatistically significant differences in the mean percent stabilityrelative to Sigma lot #3 at detergent concentration tested (FIG.11A-11D). In fact, the Sigma lot #3 shows statistically significantdifferences with all of the other lots of CHAPS at the lowest detergentconcentration (FIG. 11A). The Dojindo shows statistically significantdifferences at some detergent concentrations with Sigma lot #1 (13.44mM; FIG. 11D) and Sigma lot #2 (8.00 mM; FIG. 11C). At other detergentconcentrations, the differences in the means in the percent stabilitiesjust miss the criteria for statistical significance in the comparisonbetween the Dojindo CHAPS and the Sigma lot #1 (e.g., p-values of0.0651, 0.0862 and 0.0678 in FIGS. 11A, 11C and 11D, respectively) andthe Sigma lot #2 (e.g., p-values of 0.0855, 0.0862 and 0.0678 in FIGS.11A, 11B and 11C, respectively). Minimally, these results suggest thatthere are differences in calibrator stability performance between thedifferent lots of CHAPS tested here. These differences in stability canbe seen as early as fourteen days post-formulation.

The experimental results from the Detergent Comparison study aresummarized in FIG. 12. Even at the Day 0 time point, not all of thedetergents surveyed give a similar range of Lp-PLA2 analyte values (inng/mL) even within the titration series. In fact, some of the detergentsgive radically different analyte values across the entire titrationseries relative to CHAPS titration series with the biggest outlier beingthe detergent n-octyl-β-D-glucoside (FIG. 12). In contrast, the variouslots of CHAPS-formulated calibrators yield very similar analyte valuesat a given detergent concentration. It should be noted, though, thatLp-PLA2 analyte values, in general, incrementally decrease as a functionof increasing CHAPS concentration. Collectively, these results suggestthe following two implications: (1), the final concentration of theCHAPS detergent in the calibrator diluent formulation ultimately has asubtle effect on the exact Lp-PLA2 analyte values obtained, and, (2),switching to an alternate detergent may have a profound effect on theexact Lp-PLA2 analyte values obtained.

Example 2 Material Variation Study

Purpose: To explore the effect on calibrator stability and precision ofsubstituting different vendors' and/or different grades/lots of thevarious calibrator raw materials into the context of two differentcollections of the remaining raw materials.

Materials:

PLAC ELISA kit: P/N 90123, L/N 1012045;

Antigen: P/N 26203, L/N 1010057;

Tris base;

Standard Grade: Sigma P/N 1503, L/N 040M5439V: Test Grade: ResearchOrganics P/N 3094T, L/N A82450;

CHAPS: Standard Grade: Sigma C3023; Lot #1: L/N 018K53003; Lot #2: L/N040M5319V; Lot #3: L/N 077K530012; Lot #4: L/N 100M53082V. Test Grade:Dojindo C008; Lot #5: L/N CY783; Lot #6: L/N CY784; Lot #7: L/N CY785.

BSA: Standard Grade: Millipore “Universal”, P/N 81003; Lot A: L/N 692(current Production lot); Lot B: L/N 693; Lot C: L/N 694. Test Grade:Millipore “Diagnostic”, P/N 82045; Lot D: L/N 452; Lot E: L/N 454; LotF: L/N 453.

DTT: Standard Grade: Sigma P/N D0632, L/N 031M1753V. Test Grade:BioVectra P/N 1370, L/N 37383;

NaCl: Standard Grade: Sigma P/N 59888, L/N 040M02279V; Test Grade:Research Organics P/N 0926S, L/N Z80586;

Water: Standard Grade: JT Baker P/N 4218, L/N J50E00; Test Grade: RiccaP/N 91901, L/N 1103234

Glycerol: Standard Grade: EMD P/N GX0856, L/N 50242049; Test Grade:Research Organics P/N 9580G, L/N B90864

ProClin 300: Standard Grade: Supelco P/N 48914, L/N LB82798; TestCondition: Absent from formulation

Hydrochloric acid, Mallinckrodt, P/N 2062-46, L/N 149028;

Millipore Steriflip Express Plus, P/N SCGP00525, L/N MPSF006562;

Experimental Procedures:

Experimental Plan: Two calibrator formulation groups of raw materialswere established: one with a standard group of manufacturing rawmaterials and one with a test group candidate raw materials of adifferent grade and/or from a different vendor. The test group consistedof raw materials sourced either from potential alternate vendors of rawmaterials that had showed promise in earlier experiments (e.g., DojindoCHAPS) or from vendors of raw materials with claims of exceptionallyhigh purity (e.g., ultra-pure grade or diagnostic grade). Individual rawmaterials were systematically tested in each of two contexts: thestandard group of raw materials and the test group of candidate rawmaterials. In addition to the dry raw materials obtained as salts orpowders, two grades of water were surveyed in this comparison study(HPLC grade and USP, Ph. Eur. grade). In some cases, certain candidatecritical raw materials (CHAPS and the BSA) were evaluated by screeningmultiple lots of each grade of raw materials against both raw materialgroups. The one exception to the alternate sourcing of the raw materialswas the Proclin-300. As there is only one vendor, the variable testedwas the presence/absence of this preservative in the context of both thestandard and test groups of the other raw materials. In total,thirty-six different calibrator diluent formulations reflecting a singlesubstitution of each raw material were created. Each formulation wasspiked with antigen at a single analyte concentration were tested in along-term refrigerated stability study.

Experimental Details. Concentrated stock solutions were created for thetwo groups of the following raw materials: iris base (1.00 M), CHAPS(0.050 M), DTT (1.00M), sodium chloride (5.00M). All the raw materialsin the standard group were formulated using the Ricca USP grade water,and all the raw materials in the test group were formulated using theJTBaker HPLC grade water. Proclin300 was added neat, as needed. Forpurely technical reasons, the appropriate grade/lot of BSA was added aspowder to each formulation. Thirty-six reactions were systematicallyassembled as two separate master-mixes from the raw material stocks ((5×and 1.25×, analogous the formulation work described)). An appropriatevolume of the designated grade of undiluted glycerol was added to eachof the thirty-six formulations. After mixing, each formulation wasfiltered using a 50 mL Millipore Express Plus filtration unit followedby the addition of the recombinant protein Lp-PLA2 to a final analyteconcentration of approximately 250 ng/mL, as described. The calibratorformulations were put on stability at refrigerated temperature. The FGcalibrators were split into two sets, one stored at refrigerationtemperature and one stored frozen at −70° Celsius. Stability timepointswere taken on Day 0, Week 1, Week 2, Week 4, Week 6, Week 8, Month 4,Month 5, Month 6, Month 7, and Month 9 using the PLAC ELISA kit.Specific activity of the DTT was calculated using a quantitativesulfhydryl assay with free cysteine as a standard and following themanufacturer's recommended instructions (Pierce kit #22582; see LNB0555-108 to 0555-113).

Results

This long-term stability study is raw material component swapping studyin which each raw material used in the calibrator diluent formulation issourced from two different vendors and/or from two different reagentgrades (with the exception of the Proclin-300 preservative, in which thevariable is presence/absence). In many cases, the raw materials are oftwo different grades (FIG. 13), with one being a standard grade (RedTeam) and, typically, a test grade (Blue Team) being a high-puritycompeting raw material. The “Red Team” of raw materials used all thestandard grade of raw materials with the exception of the water. Acalibrator diluent used a pharmaceutical grade of water known as USPgrade. A USP/Eur. Ph.-certified, GMP grade of water was sourced fromRicca for use as a raw material in the standard grade combination. In2011, a grade of water known as HPLC grade was sourced from JT Baker(MSS-10107) and replaced house de-ionized water used in manufacturingstarting with ELISA kit lot number 1012111. This HPLC grade of water wasused as part of the test grade of raw materials. In addition to thewater grade comparison, the Tris buffer, CHAPS, DTT, sodium chloride andglycerol were sourced from two different vendors. The BSA was sourcedfrom the standard vendor, but two different grades were compared,Universal grade and Diagnostic grade. In addition, analytical testingwas performed on the specific activity of each grade of DTT afterformulation into calibrators to show equivalence. The mean specificactivity, relative to the free cysteine standard curve, for eachvendor's DTT was calculated on Day One, post-formulation (Sigma DTT:mean [+/−STDEV]=0.404+/−0.108 mM; BioVectra, mean[+/−STDEV]=0.460+/−0.047 mM; n=18 formulations each vendor's DTT). Inthe case of the CHAPS and BSA, at least three lots of each grade wereincluded in the study. The individual lots of CHAPS used in the standardformulation and test formulation were Sigma lot 1 (lot #018K53003) andDojindo lot 7 (L/N CY785), respectively. The individual lots of BSA usedin the standard formulation and test formulation were Universal gradelot A (lot #692, a production lot) and Diagnostic grade lot F,respectively. The experimental design consisted of systematicallysubstituting individual raw materials from a particular grade/lot fromone set of raw materials into the other collection of raw materials.

The percent stability for each of the thirty-six calibrator diluentformulations for first nine months of timepoints is shown in FIG. 14.Percent stability was calculated based on analyte values establishedusing finished good calibrators that were frozen prior to initiating thestudy. The provisional 97%-103% calibrator stability specification isshown by the red hatched lines with the target stability of 100%relative to Day 0 shown as a green dashed line (FIG. 14). The “Red Team”of raw materials is Condition #1 and the Blue team of raw materials isCondition #35. Representative examples of the substitution of individualgrades/lots are shown in Conditions #2 and #3: The Tris buffer from thevendor Research Organics is substituted into the context of the RedTeam's remaining raw materials in Condition #2, and the Tris buffer fromthe vendor Sigma is substituted into the context of the Blue Team'sother raw materials in Condition #3 (FIG. 14). Different lots of CHAPSraw materials are substituted into the Red Team and Blue Teams remainingraw materials in Conditions #4-9 and Conditions #10-15, respectively.Different grades/lots of BSA raw materials are substituted into the RedTeam and Blue Teams remaining raw materials in Conditions #16-20 andConditions #21-25, respectively. Similarly, substitution of differentlots of DTT, sodium chloride, water, glycerol and ProClin300(presence/absence) are shown for the remaining conditions (i.e.,Conditions #26-34, #36)

The best performing formulation of these thirty-six calibratorconditions is Condition #5 with respect to achieving the provisional97%-103% calibrator stability specification for the majority of theeleven timepoints (Compare Condition #5 to Condition #1, Condition #4,and Conditions #6-9; FIG. 14). Condition #5 is part of the battery ofCHAPS lot substitutions formulated in the context of the Red Team's rawmaterials. Notably, the calibrator formulation Condition #5 has thesubstitution of CHAPS lot #3 (Sigma C3023, L/N 077K530012) for CHAPS lot#1 (Sigma C3023, L/N 018K53003) in the calibrator diluent.

Other well-performing formulations of these thirty-six calibratorconditions were Condition #4, Condition #9 and Condition #28. Condition#4 and Condition #9 are part of the series of CHAPS raw material survey,substituting CHAPS lot #2 (Sigma C3023, L/N 040M5319V) and CHAPS lot #7(Dojindo C008, L/N CY785), respectively, for CHAPS lot #1 (i.e., SigmaC3023, L/N 018K53003 found in Condition #1; FIG. 14). These experimentalresults indicate that the substitution of one lot of CHAPS for anotherlot of CHAPS in an otherwise identical formulation can result in adramatic difference in long-term stability performance. Taken together,these results strongly suggest that the choice of CHAPS lot is acritical raw material in the calibrator diluent formulation in somecases.

A gauge analysis shows that the overall trending of the long-termstability results as a function of calibrator condition (FIG. 15A). Inaddition to showing the anticipated decrease in calibrator stabilityover time (FIG. 15B), the gauge analysis also showed the trending oflong-term stability as a function of the various raw material used(FIGS. 16A-16C and 17). Among the Red Team collection of raw materials,the previously-mentioned Conditions #4, #5, #9 and #28 are among thebest performers with a majority of their stability timepoints fallingwithin the 97%-103%% specification. Among the Blue Team collection ofraw materials, Condition #13 is the best formulation condition forachieving the 97-103% specification (e.g., compare Condition #13 toCondition #35; FIG. 14 and FIG. 15A). The best performer, Condition #13,is part of the CHAPS substitution series for the Blue Team's collectionof raw materials, and it substitutes CHAPS lot #4 (Sigma, C3023, L/N100M53082V) for CHAPS lot (Dojindo C008, L/N CY785). In general, thestability follows a very similar trending for the CHAPS lots across bothcollections of raw materials (compare Conditions #4, #5, #6, #7 and #8to Conditions #11, #12, #13, #14, #15, respectively; FIG. 14). The gaugeanalysis indicates that CHAPS lot #4 yielded the optimal results withrespect to long-term stability trending across the entire experiment(FIG. 16A).

Two different grades of bovine serum albumin (BSA), the standardproduction “Universal Grade” and the “Diagnostic Grade”, from Milliporewere also surveyed in this raw material comparison study (three lotseach; compare Conditions #1, #16-20 in the context of the Red Team toConditions #21-25 and Condition #35 in the context of the Blue Team,respectively; FIG. 14). Similar to the CHAPS trending (section 8.2.5),the stability follows a very similar trending for the BSA lots acrossboth collections of raw materials (compare Conditions #16, #17, #18, and#19 to Conditions #22, #23, #24, and #25, respectively; FIG. 14). Thegauge analysis indicates that BSA lot “A” (Universal Grade, L/N 692) andBSA lot “E” (Diagnostic Grade, L/N 454) yielded the optimal results withrespect to long-term stability trending across the entire experiment(FIG. 16B).

An interaction analysis indicates that there is likely to be someinteraction between the CHAPS and BSA with regard to stabilityperformance. The CHAPS lot #1 appears to be much more sensitive tosubstitutions of BSA lot whereas the CHAPS lot #7 seems more robust toBSA lot substitutions (Compare the encircled data points in FIG. 16C). Asimilar trend can be observed when the individual lot of CHAPS #2-#6 arecompared between BSA lots A and lot F (FIG. 16C). This result suggestthat there could be a need for a material qualification for BSA (similarto TM-008) when switching lots of CHAPS lot to account for potentialinteractions between CHAPS and BSA in the calibrator diluent.

The effects of varying the lots of CHAPS and BSA relative to each otherin this study are also shown in FIG. 17. Comparing formulations acrossand within a particular row indicate the effects of substituting lots ofCHAPS while keeping the BSA lot number constant. Comparing formulationsup and down a given column indicate the effect of substituting lots ofBSA while keeping the CHAPS lot number constant. In general, CHAPS lots#3 give the best stability with BSA lot A, whereas CHAPS lot #4 give thebest stability with BSA lot E (see solid orange boxes in FIG. 17). Ingeneral, BSA lot E seems to give the best stability with both CHAPS lots#1 and #7 (see hatched orange boxes in FIG. 17). In some comparisons(e.g., within BSA “A” comparison with CHAPS lot #1 and CHAPS lot #7;purple hatched boxes in FIG. 17), the raw materials used in theformulation of the Blue Team of calibrators gave higher percentstabilities, perhaps as a result of the different grade of water used informulation.

A comparison of the remaining raw material substitutions with regard tolong-term stability are shown in FIG. 18A-18F. The effects of theindividual raw materials water, Tris buffer, sodium chloride, DTT, waterand glycerol all appear to co-vary with respect to whether they are onthe Red Team or the Blue Team rather than by vendor per se (FIG.18A-18E). JMP modeling of the data set resulted in effectors with higherthan allowable Variance Inflation Factors (VIF; data not shown). As aresult of the nature of the confounding variable with the watersituation, other co-varying raw material effectors (e.g., the glycerolvendor; FIG. 19), that track with the Red/Blue team show similar gaugeanalysis responses (FIG. 18A-E). In fact, substitution of the standardEMD glycerol seems to improve the performance of the Blue Team of rawmaterials (compare Condition #32 to Condition #1, FIG. 14), and thereciprocal analysis of substituting the test glycerol into the contextof Red Team shows worse performance (compare Condition #33 to Condition#35, FIG. 14). This may demonstrate an important role for glycerol inmaintaining calibrator stability. The grade of glycerol used in thecalibrators may deserve some attention. Another potentially confoundingvariable is the water grade due to the fact that the water was used toformulate all the stock solutions within a given set of raw materialsand makes up ˜70 v/v of the total reaction volume. Experiments arecurrently in progress that will address more directly the effects, ifany, of substituting different grades of water.

On the other hand, the presence or absence of the ProClin-300 shows adifferent trending pattern (FIG. 18F) relative to the other co-varyingraw materials. The absence of ProClin-300 was tested in both the contextof the standard raw materials for both the Red Team and the Blue Team.Condition #36 was the Blue Team of raw materials without any ProClin-300added, and it was clearly the formulation condition with the worstshort-term and long-term stability of the thirty-six calibratorformulations (FIG. 14 and FIG. 15A).

The precision of the thirty-six formulations was for each of thethirty-six calibrator diluent formulations for first nine months oftimepoints, including the Day 0 time point, is shown in FIG. 20. The %CV of each of the twelve timepoints (n=2 replicates/time point) areplotted temporally and as a function of formulation condition (FIG. 20).The grand mean of all the % CV measurements for the indicated timepointsacross all formulations was 1.89% (FIG. 22A). While the grand mean ofall the % CV measurements was excellent, there were clear differences inthe individual mean % CV's between the thirty-six formulations as wellas the standard deviations of the twelve individual CV's measurementsfor the thirty-six formulations. The gauge analysis shown in FIG.21A-21E shows the individual breakouts for the trending by formulationcondition, day of the study, and selected effectors (CHAPS lot, BSA lot,CHAPS/BSA interactions). Conditions #7 and Condition #22 showed the bestoverall precision (i.e., lowest % CV's) at 1.19% and 1.20%, respectively(FIG. 21A and FIG. 22A). However, Condition #22 had the best overallstandard deviation of the twelve individual CV's measurements at 0.55%,which was a full 1.00% improvement over the average for all thirty-sixformulations (FIG. 22A and FIG. 22B). Condition #31 had the second beststandard deviation for the twelve individual CV's measurements at 0.90%,a 0.65% improvement over the average for all thirty-six formulations(FIG. 22A and FIG. 22B). When precision was trended as a function oftime, only Day 45 showed noticeably worse precision than the othertimepoints (FIG. 21B)

Of the raw materials surveyed in this study, only the CHAPS and BSA lotsshowed differential effects on precision (FIG. 21, FIG. 21 and data notshown). CHAPS lots #3, #4 and #7 all had better % CV's than the grandmean % CV (FIG. 21C), and BSA lots B, C, and D all had better % CV'sthan the grand mean % CV (FIG. 21D). An interaction analysis of the[CHAPS lot*BSA lot] for the standard deviation of the twelve individualCV's measurements indicates that the optimal material variation forprecision is the combination of [CHAPS lot #7*BSA lot B], a combinationcorresponding to Condition #22 (FIG. 21E). Swapping out the CHAPS lot(compare Condition #22 to Condition #16; FIG. 21A) or the five other BSAlots show these synergistic effects (compare to Condition #22 toConditions 21, 23, 24, 25 and #35; FIG. 21A; for descriptive statistics,see FIG. 22B).

Taken together, these results suggest that different raw materialcombinations can have a positive effect on calibrator precision, and themain driver of optimal calibrator precision appears to be thecombination of the particular CHAPS detergent lots and BSA lotsutilized. In addition to a synergistic effect on stability (Section8.3.7; FIG. 16C), these two effectors seemingly work in concert to havea synergistic effect on precision as well (FIG. 21E).

Example 3 Response Surface Design DOE Study

Purpose: To assess in detail the effects of varying the concentrationsof selected raw materials on calibrator performance in the context of adesigned experiment.

Materials:

PLAC ELISA kit, P/N 90123, L/N 1102163;

Antigen: P/N 26203, L/N 1010057;

Tris Base: Sigma P/N T1503, L/N 031M5413V;

CHAPS: Sigma P/N C3023, L/N 018K53003;

BSA: Millipore P/N 81003, L/N 692 (current Production lot);

DTT: Sigma P/N D0632, L/N 031M1753V;

NaCl: Sigma P/N 59888, L/N 040M0225V;

Water: JT Baker P/N 4218, L/N J45E01;

ProClin 300: Supelco P/N 48914-U, L/N LB82798;

EDTA: Fluka P/N 003777, L/N BCBD4995V;

Millipore Steriflip Express Plus, P/N SCGP00525, L/N MPSF006562;

Experimental Procedures:

Experimental Plan. An earlier development report, DR-00133, describedthe results of a two-level full-factorial design for four potential rawmaterial effectors of calibrator stability in a short-term refrigeratedstability study. The four effectors were the pH of Tris-HCl buffer,[7.40, 8.00]; NaCl, [0.154 M, 2.857 M]; CHAPS concentration, [0 mM, 4.76mM]; and, EDTA concentration, [0 mM, 0.5 mM]. In addition, a fifth,categorical effector was also surveyed: Reducing Agent Identity [None,DTT 0.95 mM, TCEP 0.95 mM]. The standard calibrator diluentconcentrations are underlined, and the optimal conditions trendedtowards the underlined (i.e., standard) concentrations. In addition tothe high salt concentration, the inclusion of both the standardconcentrations of both DTT and CHAPS was particularly important forcalibrator stability. The role of pH in short-term calibrator stabilitywas less clear and seemingly context-dependent.

In this response surface experimental design, the sodium chloride andreducing agent identity were kept constant, and the concentrations ofthe protons (i.e., pH), buffer concentration, DTT and CHAPS weresurveyed using an optimization technique known as a response surfacedesign (specifically, the RSD is a rotatable central composite design).Because central composite designs contain design points from a two-levelfactorial design (augmented by center points and numerous axial points),they are useful for sequential experimentation. With theseconsiderations in mind, the midpoint and factorial points representedstandard raw material concentrations, and the axial points representedopportunities to screen for raw material concentrations that result ineither stability improvements or “test-to-failure” outcomes.

The DTT and CHAPS concentrations were explored both above and belowtheir standard concentrations, 0.95 mM and 4.76 mM, respectively. Thehigher concentrations surveyed were performed with the possibility inmind of enhancing calibrator long-term stability. Conversely, the lowerconcentrations of each raw material were surveyed in an attempt to “testto failure”.

The pH of the calibrator diluent reagent was surveyed within arelatively narrow titration window [7.80, 7.87, 7.95, 8.05, and 8.18] aspart of a targeted optimization effort focused on improving long-termstability.

A survey of higher Tris buffer concentrations was performed to screenfor potentially beneficial effects on long-term stability.

Experimental Details. Concentrated stock solutions were created for thetwo groups of the following raw materials: CHAPS (0.100 M), DTT (1.00M),sodium chloride (5.00M) and Proclin-300 (10% v/v). The Tris baseconcentrated stock solutions (1.00 M) were created at five differentpH's (8.45, 8.25, 8.15, 8.07 and 8.00) to mimic a process in which thestarting pH is intentionally set 0.20 pH units more alkaline than thedesired final pH prior to the addition of the BSA. All the raw materialswere formulated using the JTBaker HPLC grade water. For purely technicalreasons, the Millipore BSA (lot 692) was added as powder to eachformulation. Briefly, nine separate Buffer A mastermixes, representingthe nine buffer concentration/pH combinations, were equilibrated withthe common BSA/ProClin-300 components in an isotonic solution.Separately, twenty-six individual Buffer B master-mixes were formulatedwith the DTT/CHAPS components in a high-salt, buffered solution. A pHadjustment was performed with hydrochloric acid/sodium hydroxide toachieve the intended final pH for each of the fifty-two conditions.After mixing, each formulation was filtered using a 50 mL MilliporeExpress Plus filtration unit followed by the addition of the recombinantprotein Lp-PLA2 to a final analyte concentration of approximately 250ng/mL, as described in the section above. The calibrator formulationswere put on stability at refrigerated temperature. Stability timepointswere taken on Day 0, Day 3, Day 8, Week 2, Week 4, Week 8, Month 3,Month 4, Month 5, and Month 6 using the PLAC ELISA kit.

Results

Response Surface Design DOE Study, Six Months

Previously, a full factorial design DOE was described in which extremelevels of selected raw materials concentrations in the calibrator werescreened, including buffer pH, sodium chloride concentration, and theinclusion/exclusion of CHAPS detergent and choice of reducing agent (ornone at all). This Response Surface Design DOE study was a follow upstudy with the goal of optimizing the concentrations of selectedstandard raw materials in the calibrator diluent, including CHAPS, thereducing agent DTT, Tris buffer concentration and Tris buffer pH.

A response surface design is a type of designed experiment that uses asecond-degree polynomial model to obtain an optimal response. A centralcomposite design is a particular type of response surface design thatcontains an imbedded factorial design with center points that isaugmented with a group of “star points” (also known as axial points)that allow an estimation of curvature (FIG. 23). The star points are atsome distance from the center based on the properties desired for thedesign and the number of factors in the design. The star pointsestablish new extremes for the low and high settings for all factors andare surveyed in conjunction with the midpoint concentrations of theother effectors. The presence of these axial, or “star points”, is oneof the characteristics that distinguishes the central composite designfrom other types of response surface designs, such as a “Box-Bhenken”design. A full description of the response surface design can be foundin the legend to FIG. 23.

A total of twenty-six formulation conditions were surveyed in thisexperimental design including a duplication of the center pointformulation. Sixteen formulation conditions are contributed by therequirements of the full factorial design (24), which are represented bythe sixteen vertices of the factorial design (i.e., in four dimensions).The remaining eight conditions are represented by the two axialpositions of each of the four raw materials. The net result is fiveconcentrations are tested for each raw material in various contexts ofthis type of design (FIG. 23). A full description of the individualformulation conditions is described in FIG. 24.

The long-term stability results are shown for multiple timepoints, takenover the course of six months duration (FIG. 25). Stability wascalculated based on optical density measurements relative to the samemeasurement on Day 0 of the study. Several of the conditions showedexcellent stability depending on the metric used for stability.Condition #3 had the lowest mean difference in percent stability for thenine timepoints relative to achieving the 100%+/−3% specification at1.745% (FIG. 26, FIG. 24). This is expressed as the mean of the absolutevalue of the percent stability difference for each of the individualtimepoints relative to 100% stability in FIG. 26. Condition #1 had asmallest standard deviation (0.982%) and the lowest upper 95% confidenceinterval (+/−3.063%) for the nine timepoints' percent stabilities. Thisformulation had the highest pH surveyed (i.e., pH 8.18) and encoded byan axial point in the design (FIG. 26, FIG. 24). Condition #25 was theonly formulation to have eight out of the nine timepoints meet the+/−3.0% specification.

The three formulation conditions that demonstrated the worst stabilitywere Conditions #8, #11 and #12. These three conditions did not scoreeven a single data point from any of the nine timepoints within the+/−3.0% specification (FIG. 26). Clearly, the worst performer wasCondition #12, the axial concentration with lowest CHAPS detergentconcentration surveyed (namely, 0.90 mM CHAPS, or about 19% of thestandard concentration) with the lowest mean stability of the twenty-sixconditions (FIG. 24) and the stability got worse over time (FIG. 25).The second worse formulation for long-term stability was condition #11,the axial concentration with the lowest DTT concentration surveyed(namely, 0.05 mM DTT, or about 5% of the standard concentration).Condition #11 had the second lowest mean stability (FIG. 24) of thetwenty-six conditions. Interestingly, the stability of the Condition #11formulation did not appear to worsen over time (FIG. 25), as there wasjust an apparent 10% drop-off in stability relative in the first tendays of the study to the initial (day 0) time point.

The trending of the gauge analysis for the four effectors studied hereshows concentration-dependent effects for two of the raw materials.Consistent with the results of the “Detergent Comparison Study” (e.g.,see FIG. 10D), there is noticeable drop in stability when the CHAPSconcentration is lowered from the standard concentration of 4.76 mM to2.83 mM and an even sharper drop in stability when the CHAPSconcentration is lowered to 0.90 mM (FIG. 27A). The stabilityperformance seems to plateau at CHAPS concentrations above 4.76 mM, atleast within the six month timeframe analyzed here. There is also anoticeable drop in stability when the DTT concentration is drops below0.35 mM to the next surveyed concentration of 0.05 mM (FIG. 27B). Thestandard concentration used is 0.95 mM. There may be a peak in thestability response to DTT somewhere around the midpoint concentration ofDTT tested (0.65 mM; FIG. 27B). Neither the Tris buffer pH (FIG. 27C)nor the Tris buffer concentration (FIG. 27D) demonstrated any noticeabletrending within the six month timeframe analyzed here. There was someday-to-day variability within a total range of approximately 5% in themeasured OD values relative to Day Zero (FIG. 27E), but the gaugeanalysis of the measurements from day-to-day did not follow anydiscernible trend.

The data was analyzed using the JMP “Response Surface” functionality tofit the data to a model which included the four raw materialconcentration and time as effectors. The nine points after Day Zero weremodeled using time as one of the effectors and eliminating the conditionwith the lowest concentration of CHAPS (0.90 mM) from the model too muchsensitivity was lost when it was included. The model was refined byremoving effectors and second-degree interactions sequentially that werenot statistically significant (p value <0.05) as described in the legendto FIG. 28A-28C.

The modeling using of the data should be interpreted with severalcaveats. The model had both a marginal R-squared value (0.246) and astatistically significant lack of fit (p value <0.0001; FIG. 28A)

On the other hand, the Analysis of Variance (ANOVA) for the model wasstatistically significant (p value <0.0001) and the F-ratio wasacceptable (F Ratio=7.82; FIG. 28A)).

Numerous parameter estimates, including several quadratic interactionsshowed statistical significance and excellent VIF's (FIG. 28B):

Time, p<0.0001

[DTT*DTT], p<0.0001

CHAPS, p=0.0040

[Time*Time], p=0.0089

[pH*Time], p=0.0132

[DTT*CHAPS], p=0.0302

[pH*CHAPS], p=0.0467

The prediction profiler functionality in JMP was utilized to predict theoptimal raw material concentrations for the statistically significanteffectors. The Buffer pH was kept in the refined model even though itwas not statistically significant itself because pH showed astatistically significant quadratic interaction with the effector, Time.

Buffer pH trends toward pH 8.05 (FIG. 28C, panel 1). This pH fallswithin the standard pH specification of 7.95-8.05 for the calibratordiluent formulation. The quadratic interaction of buffer pH with time(i.e., [pH*Time]) was of modest statistical significance (p=0.0132)

DTT concentration trends toward 0.70 mM (FIG. 28C, panel 2). Thestandard concentration used in the formulation is 0.95 mM. Notably, theDTT itself was not statistically significant, but the quadraticinteraction of [DTT*DTT] showed excellent statistical significance(p<0.0001). In addition to the quadratic interaction of DTT with itself,the DTT concentration also showed a quadratic interaction of modeststatistical significance (p=0.0302) with the detergent CHAPS (i.e.,[DTT*CHAPS]).

The CHAPS concentration trends toward 6.69 mM (FIG. 28C, panel 3) andshowed good statistical significance (p=0.0040). This concentration isabout 40% higher than the standard concentration of 4.76 mM used in thecalibrator formulation. In addition to the above-mentioned interactionwith the DTT, the CHAPS detergent also showed a quadratic interaction ofmodest statistical significance (p=0.0467) with the effector, pH (i.e.,[pH*CHAPS]).

The relationship between the main effectors in the response surfacedesign (CHAPS, DTT and pH) are parsed diagrammatically in FIG. 29. Theeffects of the test-to-failure, low concentrations (axial) of CHAPS andDTT show obvious deleterious effects on stability (see red box and theblue box, respectively, in FIG. 29). The deleterious effects of thesecond lowest concentration of CHAPS tested (i.e., 2.83 mM) wereselectively observed for the conditions at high DTT concentration (0.95mM DTT, and the conditions most similar to the those surveyed in theDetergent Comparison Study) and not low DTT concentration (0.35 mM DTT;compare hatched pink box to the solid pink box, respectively, in FIG.29). At higher CHAPS concentrations, there is no differential stabilityobserved between the high and low DTT concentrations (compare the traceswithin the hatched and solid green boxes, respectively, in FIG. 29). Athigher CHAPS concentrations, the pH 8.05 subtly out-performs the pH 7.87conditions when the purple traces are compared to the red tracesresiding within the two green boxes in FIG. 29. On the other hand, atthe low CHAPS concentration (i.e., 2.83 mM), the pH 7.87 appear to farebetter in stability than the pH 8.05 (compare the red traces to thepurple traces within the solid and hatched pink boxes, respectively, inFIG. 29). Consistent with the JMP modeling, very good stability is alsoshown by Condition #1 (the orange trace in the orange box in FIG. 29),the axial condition representing the highest (most alkaline) pH surveyedin the context of the midpoint concentrations for the other three rawmaterials. These interactions between [pH*CHAPS] and [DTT*CHAPS] werepredicted by the JMP modeling (Section 8.4.7.3), and these differentialeffects on stability can be visualized using the type of diagram shownin FIG. 29.

The precision of the twenty-six calibrators was also analyzed as afunction of raw material concentration. Imprecision was calculated foreach condition at each of the ten timepoints, n=2 replicates per timepoint. Overall, the precision across the experiment was very good, witha grand mean % CV of 2.22% (FIG. 30A). Condition #3 and Condition #25had the best overall precision with average % CV's of 1.12% and 1.15%,respectively (FIG. 30B). Both these conditions utilized the 6.69 mMCHAPS concentration (FIG. 31A). Condition #17 (axial point for highbuffer concentration) had the best standard deviation of the ten % CVmeasurements at 0.78% with Conditions #3 and #25 being tied for thesecond-best measurement at 0.92% (FIG. 30B). Modeling of the imprecisiondata using JMP gave poor results (data not shown).

The imprecision of the measurements for all twenty-six conditions wasanalyzed by gauge analysis, and the mean % CV was at the grand mean % CVon Day 0, but got worse on the next two timepoints on Day 3 and Day 8(FIG. 31B). The precision measurements stabilized after about 28 daysand remained relatively consistent for the remaining five months of thestudy (FIG. 31B). Additional gauge analyses using both the mean % CV ofall the measurements (FIGS. 32A-32D) and the mean standard deviation ofthe % CV's for the ten measurements for each condition (FIG. 32F-32H)was performed for each of the four raw materials as a function ofconcentration. In general the second highest CHAPS concentration (FIG.32A), the highest buffer pH (8.18; FIG. 32C) and the highest bufferconcentration showed the best performance by mean % CV (FIG. 32D).Surprisingly, the highest concentration of CHAPS showed the worstperformance in terms of mean % CV (FIG. 32A). Using the mean standarddeviation of the ten % CV measurements as a metric, the highest CHAPSconcentration again showed the worst performance (FIG. 32E), and thehighest buffer concentration showed the best performance (FIG. 32H). TheDTT concentration showed no discernible trend by either metric (FIGS.32B and 32F).

Summary: Many of the formulation conditions surveyed here showedexcellent calibrator stability performance relative to both the assigned+/−3% stability specification as well as excellent precision. Takentogether, the combination of the JMP model fitting analysis ofstability, the gauge analysis of both stability/precision, and theconsideration of the relative performance of individual formulationconditions in both stability/precision can be used to make some generalconclusions. The JMP modeling suggests that optimal stability may beconferred as the CHAPS concentration approaches 6.69 mM and possiblesynergistic effects between the CHAPS and DTT and the CHAPS and pH. TheJMP modeling also suggests a curvature to stability response withrespect to the DTT concentration. The JMP predicted an optimal pHtrending towards pH 8.05 and the axial point for high pH (8.18) in theexperiment yielded excellent stability performance. With respect toprecision, the gauge analysis suggest a small, gradual improvement inprecision when the CHAPS concentration increased up to 6.69 mM followedby a dramatic worsening in precision performance upon further increasingof the CHAPS concentration to 8.62 mM. There is also a possibility ofprospective precision improvements being gained by increasing the pH to8.18 and/or by increasing the buffer concentration to 85 mM. Whilepromising, it should be noted that these axial buffer formulationconditions were individually tested in the context of the midpointconcentrations of the other raw materials. They were neither tested incombination with each other nor in the context of the optimal CHAPSconcentration.

Example 4 Buffer and BSA Survey

Purpose: To study the effect of various buffer compositions, pH andprocess changes as well as various BSA grade/lot substitutions oncalibrator stability and precision.

Materials:

PLAC ELISA kit: P/N 90123, L/N 1106130

Antigen: P/N 26203, L/N 1010057

Buffering agents: Tris Base solution (1.0 M solution, titrated with HClby manufacturer): Teknova, P/N T1080 (L/N 16D1001, L/N 08L1001); TrisHydrochloride salt (MW 157.6 g/mol); Sigma P/N T3253, L/N 071M5401V;Tris base (MW 121.1 g/mol); Sigma P/N T1503, L/N 031M5413V;

CHAPS: Dojindo P/N C008, L/N DC862

BSA: various grades and lot numbers screened: (1) Millipore “Universal”grade, P/N 81003. Per the manufacturer, this grade is manufactured by aproprietary heat-shock fractionation process, using caprylic acid as analbumin stabilizer. A highly consistent and widely used grade of BSApowder for diagnostic, cell culture and microbial fermentationapplications. Assay: Purity 98%-100%, IgG below detectable limits. (L/N692, L/N 693, L/N 694); (2) Millipore “Diagnostic” grade, P/N 82045. Perthe manufacturer, this grade is manufactured by a proprietary heat-shockfractionation process, and this BSA powder is treated to insureinactivation of proteolytic activity. Assay: Purity 98-100%, proteasebelow detectable limits, IgG below detectable limits. (L/N 452, L/N 453,L/N 454); (3) Millipore “Fatty Acid-Free” grade, P/N 82002. Per themanufacturer, this grade is a highly purified BSA powder extensivelytreated to remove fatty acids. This grade is manufactured by aproprietary heat shock fractionation process. Assay: Purity 98-100%,free fatty acids 0-0.2 mg/g. (L/N 120; L/N 131; L/N 134);

DTT: Sigma P/N D0632, L/N 051M1871V;

NaCl: Sigma P/N 59888, L/N 040M0225V;

Water: JT Baker P/N 4218, L/N J45E01;

ProClin 300: Supelco P/N 48914-U, L/N LB82798;

Glycerol: EMD GX0185-6, L/N 50349114;

Millipore Steriflip Express Plus, P/N SCGP00525, L/N MPSF006562.

Experimental Procedures:

Experimental Plan. The experimental plan includes five different seriesof buffer conditions regarding the form, concentration, starting pH, andfinal pH used in the calibrator diluent formulation. A survey ofdifferent grades of Millipore BSA grade is also included in the study.Series A involves using a pre-formulated solution from Teknovaconsisting of 1.00 M Tris base (titrated with hydrochloric acid) to pH8.00. Included within this series is a titration of Tris concentrationup to 125 mM in 25 mM incremental steps. Series B involves using theTris-Hydrochloride salt form of the buffer, with or without a pre-pHstep. A “pre-pH” step refers to a process step in which a starting pH isestablished following the addition of the buffer and sodium chloride tothe formulation. This pre-pH process was first implemented in thecontext of using the Tris base in the calibrator diluent formulation.Originally, the pre-pH process step was implemented to circumvent aformulation process issue in which the BSA will precipitate between pH9.0 and 8.5 as the solution becomes more acidic upon titration ofhydrochloric acid (This presumably is a consequence of the BSAundergoing a pH-dependent structural transition known as the N-Btransition). Series C involves titrating a 1.00 M Tris-Hydrochloridesolution and a 1.00 M Tris base solution against each other to achievethe indicated starting pH. Similarly, Series D involves using the Trisbase form of the buffer with a pre-pH step and three different startingpH's. Series E is the survey of three different grades and/or lots ofBSA obtained from Millipore, prepared according to a standard process. Atotal of twenty-six different formulations were surveyed in this study.Each calibrator reagent was assayed using a PLAC ELISA kit over the ninetimepoints.

Experimental Details. A concentrated master-mix (4×) of the rawmaterials (CHAPS, DTT, and Proclin-300) common to all formulations inthis study was prepared and an appropriate volume was added last to allreactions to achieve the standard final concentrations of thesecomponents. Separately, thirty individual formulations reflecting theintended material/concentration permutations for the buffer and/or BSAgrade as well as the pH variation for the buffer were prepared. In manycases, a pre-pH step was implemented to establish an initial pHfollowing the addition of the buffer and sodium chloride. Afterexecution of the pre-pH step (if necessary), the indicated grade/lot ofBSA was added and mixed. After the addition of the master-mix, the finalpH was titrated using hydrochloric acid or sodium hydroxide, asappropriate, for each of the thirty formulations. An appropriate amountof 100% glycerol is added so that the final concentrations of all rawmaterials are at their standard calibrator diluent concentrations perMP-21090. After mixing, each formulation was filtered using a 50 mLMillipore Express Plus filtration unit followed by the addition ofrecombinant protein Lp-PLA2 to a final analyte concentration of 250ng/mL as described in the Experimental Procedures. The calibratorformulations were aliquoted, and each of the thirty formulations was puton stability at both refrigerated temperature and −70° Celsius.Stability timepoints were taken on Day 0, Day 1, Day 4, Week 1, Week 4,Week 6, Month 2, Month 3, and Month 4 using a PLAC ELISA kit.

Results

Buffer and BSA Survey

A comparison of protocols for the calibrator diluent formulationrevealed several differences in the raw materials (Tris buffer, watergrade), methods (a pre-pH step) and supplies (filtration) used at thetwo CMO's. A pre-pH step is used when using the Tris base as a startingmaterial for the diluent in order to circumvent a bovine serum albuminprotein precipitation issue. The first experimental goal of this studywas to compare differences both materials and formulation processes withregard to the Tris buffer. Eighteen different permutations of Tris rawmaterials, methods, pH, and concentrations were screened for potentialeffects on calibrator stability and precision. The experimental planalso directly compares the Tris material used in the originaldevelopment report conditions directly to the current methodologies. Thesecond experimental goal was a more extensive comparison of availableMillipore Probumin BSA grades known as Universal, Diagnostic and FattyAcid-Free (for full description of different grades, see section5.1.4.5). Three lots of each Probumin BSA grade were tested in thecontext of the MP-21090 standard formulation process for effects oncalibrator stability and precision. A total of twenty-six differentformulations were screened in this study.

A schematic of the experimental design for the Buffer/BSA surveystability study is shown in FIG. 33. Conditions #1 and #2 are acomparison of two lots of the Teknova Tris buffer product that was usedin the original development report to assay stability, used at thestandard concentration. Conditions #3-#6 survey higher concentrations,in 25 mM increments, of the indicated Teknova Tris buffer lot used inCondition #2 (FIG. 33). Conditions #7-#11 utilize the hydrochloride formof the Tris buffer, comparing different buffer concentrations andsurveying different combinations of with or without the pre-pH stepand/or different starting/final pH's (FIG. 33). Condition #8 mimics acurrent process. Conditions #12-#14 utilize the hydrochloride and baseforms of the Tris buffer, titrated against each other at the indicatedpH (FIG. 33). Conditions #15-#18 utilize the base form of the Trisbuffer, each surveying different combinations of starting/final pH's(FIG. 33). Condition #18 mimics another current process. Conditions#18-#26 survey the substitution of different grades/lots of Probumin BSAinto a standard formulation raw materials/process (FIG. 33). Eachformulation was put on stability at refrigerated temperature and −70°Celsius and stability was monitored over the course of four months.

The percent stabilities (mean+/−STDEV) for each of the twenty-sixcalibrator formulations on stability at two storage temperatures(refrigerated and −70 Celsius) are shown in FIG. 34. In general, themean stabilities for all twenty-six formulations were very good at bothstability temperatures across the eight timepoints. The one formulationcondition that particularly stood out was Condition #1 stored atrefrigerated temperature with a mean stability of 100.75%+/−1.62% (FIG.34). Curiously, this high achievement was not replicated by an otherwiseidentical formulation (i.e., Condition #2; 103.06%+/−2.76%) on stabilityat the refrigerated temperature. Condition #2 utilized a different lotof Teknova buffer of an identical starting pH to Condition #1 (namely,pH 8.02).

A comparison of the percent stabilities across the initial eighttimepoints (Day 1, 4, 7, 30, 45, 60, 90, 120) for the formulations onstability at −70 Celsius and refrigerated temperature are shown in FIG.35 and FIG. 36, respectively. The gauge analysis shows some interestingtrends. The calibrators stored at the two temperatures show very similartrending for buffer composition and buffer process (reflecting smalldifferences in trend lines for pre-pH and final pH), but they showslightly different trending in response to BSA lot number and day ofassay (Compare red traces in FIG. 37A-37G to FIG. 38A-38G). Overall, thedifferences observed are relatively small in magnitude with the trendlines mostly falling within the +/−3% specification with the exceptionof the day-to-day variation.

A comparison of the imprecision across all nine timepoints (includingthe Day 0 time point) showed good congruence between the averageprecision for the twenty-six formulations across two storagetemperatures (FIG. 39). The best formulation for precision was probablyCondition #18, which mimics a current formulation raw materials/process.Condition #18 demonstrated a mean % CV of 0.86% (+/−0.66%) and 0.89%(+/−0.67%) at −70 Celsius and refrigerated temperature, respectively.Condition #17, which differs only slightly from Condition #18, was alsoimpressive, with a mean % CV of 0.84% (+/−0.95%) and 0.84% (+/−1.01%) at−70 Celsius and refrigerated temperature, respectively. Condition #11and Condition #23 also showed promising precision. Condition #11 isidentical to Condition #17 but pre-pH's the hydrochloride salt ratherthan the base form of Tris to the indicated starting and final pH's(FIG. 34). Condition #23 is identical to Condition #18 except itsubstitutes Diagnostic Grade BSA L/N 454 for the standard UniversalGrade BSA L/N 692. A full comparison′ of the precision performance atboth storage temperatures, −70 Celsius and refrigerated, for each timepoint in the first four months of this stability study can be found inFIG. 40 and FIG. 42, respectively.

The gauge analysis indicates very similar trending for the precisionresults at both storage temperatures. The trends when broken out bycondition number, time, buffer composition, pre-pH (starting pH), finalpH, buffer concentration and BSA lot number show also identical tracesfor the calibrators stored at both temperatures (Compare FIGS. 41A-41Gand FIGS. 43A-43G). The buffer composition shows little effect (FIGS.41C and 43C), but the starting pH and final pH trend toward pH 8.15(FIGS. 41D, 41E and FIGS. 43D and 43E, respectively). Bufferconcentration did not show any apparent trend (FIGS. 41F and 43F), butBSA lot number does show an apparent trend with respect to precisionperformance. The BSA trending indicates that the individual lot of BSA,rather than the grade, has the most effect on performance with respectto precision (FIG. 41G and FIG. 43G). Diagnostic Grade Probumin lotnumber #454 is particularly promising by this metric. Fatty Acid-FreeProbumin lot #120 was the worst performer by this metric.

The gauge analysis was also performed across all the calibratorformulations to look at the overall trending of both stability andprecision performance as a function of storage temperature. Thecalibrators stored at −70 Celsius trended right at −100% stabilityrelative to their Day 0 OD value whereas the refrigerated calibratorscame in slightly higher at −101%. With regard to precision, there wasvirtually no difference between the calibrators stored at differenttemperatures.

In Examples 1-4, above, a central focus of the continuous productimprovement effort is the formulation of a calibrator diluent matrixuseful for assays including (but not limited to) ELISA kit calibrationstandards. For example, the calibrators may cause ELISA kit stabilityissues if not properly managed. The studies presented here comprise aseries of experimental approaches focusing on the formulation of thecalibrator matrix and are intended as follow up studies to an initialshort-term stability study. In an earlier development report, key inputsfor maximal calibrator stability were identified: the inclusion of boththe CHAPS detergent and a reducing agent in the context of the standardbuffer conditions including the high-salt solution. In the studiespresented here, component swapping studies and a designed experimentwere used to establish a detailed understanding of the effects of keyinputs (raw material variability, raw material concentrations andformulation process) on the outputs (calibrator stability and calibratorprecision). The characterization of the effectors suggests optimaltarget concentrations of certain raw materials that may be useful for amore robust design of the calibrator matrix. This robust design plan mayutilize two tactics. The first tactic is to adjust the targetconcentrations of certain raw materials so that the output (percentstability) is less sensitive to any variability in the input (namely,raw material concentration; see FIG. 45A). A typical scenario is when aresponse surface design experiment indicates a non-linear relationshipbetween an input and an output. Reducing variation in the outputrequires re-designing the process/formulation so that the variationtransmitted to the output is minimized for a given amount of inputvariation (See FIG. 45A for a textbook example). The second tactic is toadjust the target concentration of the inputs in such a way as tominimize the variance in the output of the mean stability measurement ata given time point. Since the standard deviation of the measurements isthe square root of the variance, the implementation of this approachinvolves minimizing the coefficient of variation of the stabilitymeasurement taken at each time point. The results of these fourstability studies suggest practical ways of utilizing both tactics tooptimize and improve the performance of a calibrator matrix with respectto both calibrator stability and precision.

Regarding example 1, the detergent comparison stability study, thedetergent comparison study was a component swapping experiment in whichselected membrane detergents/CHAPS analogues were screened in ashort-term real-time stability study. A key aspect of this study is thatthe detergent concentrations chosen were normalized based on theirrespective critical micelle concentrations (CMC's), a value specific toeach detergent. With respect to maintaining Lp-PLA2 stability, thegeneral trend for the set of detergents was stabilized was maximal whenthe detergent concentration was at CMC (or higher) with a sharp drop offin stability at concentrations lower than CMC. This result stronglysuggests that micelle formation may be important for maintaining Lp-PLA2stability across the entire panel of detergents surveyed, at least insome situations. When CHAPS was studied to the exclusion of the otherdetergents, the lots of CHAPS analyzed here actually showed slightlybetter stability at sub-CMC concentrations than did the otherdetergents. The 0.595× concentration of CHAPS (corresponding to astandard [4.76 mM] CHAPS concentration in a reference calibrator matrix)showed comparable stability to the 1×CMC concentration, but thestability profile showed ˜10% drop-off at the 0.354× concentration(corresponding to the 2.83 mM CHAPS concentration).

The detergent comparison study also demonstrated that there isdifferential calibrator stability observed when using different lots ofCHAPS detergent. In a comparison of four different lots of CHAPS fromtwo vendors, statistically significant differences in stability wereobtained using a Student's t-test even within the timeframe a 30-dayshort-term stability study. Notably, the difference in stability betweenthe Dojindo lot of CHAPS (lot number CT717) and the Sigma lot #3(BioXtra, lot number 18K530041V) yielded a statistically significantdifference at every CHAPS concentration tested. Given that standardconcentrations of other raw materials were used in this study, theseresults suggest the possibility that differences in stability as afunction of detergent concentration can be observed even in a relativelyshort timeframe. It should be noted, though, that the differential inpercent stability observed with some of these lots of Sigma CHAPS(namely, lots 018K53003 and 040M5319V) is of a greater magnitude thanthat observed in subsequent stability studies with the same two lots ofSigma CHAPS in the Mix-and-Match Study. On the other hand, the singlelot of Dojindo CHAPS tested demonstrated good stability at the standardCHAPS concentration and higher when tested using the same pre-formulatedmaster-mixes of the remaining raw materials common to each formulation.The subsequent studies reported here used the BSA grade from the vendor(Millipore Probumin Universal grade) as well as certain raw materials(GMP grade hydrochloric acid) of a potentially higher quality. The roleof individual lots of CHAPS detergent was explored in more detail in theMix- and Match Experiment with a more complete collection of standardraw materials.

A variety of other detergents were screened in the Detergent Comparisonstudy to assess the feasibility of using alternate detergents tostabilize Lp-PLA2. The two CHAPS analogues, CHAPSO and sodium cholate,showed promising short-term stability results. In contrast, two othertwo CHAPS analogues, BIGCHAP and deoxy-BIGCHAP, were less promisingalternatives. The n-octyl-β-glucoside showed some promise with itsperformance in this initial screen, and it was the detergent used in thedetermination of the structure of Lp-PLA2 by x-ray crystallography. Then-octyl-β-maltoside showed less promising short term stability indicatedby a sharp drop-off in percent stability between the Day 14 time pointand the Day 0 time point. The MEGA-8 may be more difficult to use inpractice as it requires a relatively high detergent concentration (˜30mM) for effective protein stabilization. An additional complication isthat the current purification scheme utilizes CHAPS throughout theprocess for isolating recombinant Lp-PLA2 antigen. Switching detergentsin the calibrator formulation may require additional validation studies,supplier qualification by QA and/or additional Clinical/Regulatorystudies. With these considerations in mind, the remainder of the studiesreported here focused exclusively on the CHAPS detergent.

Regarding example 2, the Material Variability Stability Study, thisstudy focused on a strategy of individually substituting the entirebattery of raw materials from various grades/vendors into two separatebase formulations of raw materials. The base formulations comprised twocollections of raw material groupings: a standard grade from currentvendors and a test grade of alternative raw material vendors. Inaddition to the substitution of different raw materials of differentgrades or from different vendors, multiple lots of the CHAPS (fromSigma/Dojindo vendors) and BSA (from Universal/Diagnostic grade) werealso surveyed. These two raw materials are likely candidates to havesignificant lot-to-lot variability based on both the well-characterizedheterogeneities in lots of BSA and the reported trace contamination ofCHAPS preparations with precursor molecules. The contaminatingprecursors of detergents have been shown to completely solubilize intothe interior of the detergent micelles, affecting both micelle size andaggregation number. In many cases, the trace contamination of thesurfactant preparations with precursor molecules that “show strongersurface activity than that of the main component”, and these “highlysurface-active contaminations can affect significantly properties of thesystem investigated”. The BSA preparations are also subject to a numberof well-characterized heterogeneities including variable proportions ofdimers and higher oligomers (polymerization/aggregation), mixeddisulfide bond formation, IgG contamination, fatty acid contamination,lot-specific protein contaminants. Physico-chemical studies suggest thatBSA can adopt different pH-dependent three-dimensional shapes insolution, assuming a prolate ellipsoid (cigar-shaped) conformation atslightly alkaline pH (i.e., 8.3) versus assuming a heart-shapedconformation at neutral pH. Knowledge of which analytical specificationsand/or which process variables of the BSA actually affect productperformance may be important.

The Material Variation stability study used essentially all standard rawmaterials for the one grouping of raw materials that is referred to asthe “Red Team”. Care was taken to use a particular Universal grade BSA(P/N 81003, L/N 692) as part of the standard grouping of raw materials.The exception is that pharmaceutical grade water (Ricca P/N 9109;USP/Ph. Eur. grade) was used in the standard grade collection of rawmaterials to mimic the manufacturing protocol used previously. The testgrade of raw materials used the HPLC grade water (JTBaker P/N 4218). TheBlue Team of raw materials consisted of reagents sourced from differentvendors and/or different grades. The effects of these materialvariations on performance were studied by measuring the responses onboth stability and precision using variability gauge analysis.

Main effectors on stability were the lot number of CHAPS and BSA, andthese two effectors may interact synergistically. In general, the RedTeam, which comprises the standard collection of raw materials, showedbetter stability than the Blue Team of test raw materials within thedesignated 100%+/−3% specification used in this analysis. A comparisonof the individual formulation conditions within the Red team of rawmaterials alone indicated CHAPS lot #2 (L/N 040M5319V) and lot #3 (L/N077K530012) showed the best stability profile across all time points inthis study. Within a comparison of the individual formulation conditionswithin the Blue team of raw materials, these CHAPS lot #4 (Condition #13using CHAPS L/N 100M53082V) yielded the best stability results, with thecaveat that all the Blue Team formulation conditions showed“over-recovery” relative to 100%. Averaged across both the Blue and RedTeams of raw materials, the CHAPS condition #4 showed the best resultsin the gauge analysis across these two conditions. The results with theCHAPS detergent suggest that it is a critical raw material in thecalibrator diluent formulation and some analytical specification orincoming quality control metric may need to be established that ispredictive of calibrator stability. The BSA lots also showed dramaticdifferential effects on stability, with the Millipore Universal BSA “A”(lot 692) and the Millipore Diagnostic Grade “E” (lot 454) demonstratingthe best results. It should be noted, however, that the Diagnostic GradeBSA “E” showed the greatest difference in stability in the interactionanalysis when Red and Blue teams of raw materials are compared (see FIG.16C). The other ten combinations of CHAPS/BSA seem to track prettyclosely to each other when comparing performance across the Blue and Redteams. Both the CHAPS and BSA demonstrate lot-specific differences, andthey do not necessarily correlate with either raw material vendor/gradeor any other obvious vendor-provided product specification.

Given the almost identical trending of most of the other raw materials,other raw materials were considered as potential effectors for the Blueteam showing more “over-recovery” relative to day Zero compared to theRed team across the study. Two other raw materials, the water grade andthe glycerol grade, were considered as candidate effectors of stabilityas well. Another raw material that is a good candidate for having adeleterious effect on stability is the glycerol. The standard grade ofglycerol showed the best performance and was used in the Red team ofreagents. Assuming there is little lot-to-lot variability in a standardvendor glycerol, the glycerol is not likely to be a root cause ofcalibrator instability. However, if there is lot-to-lot variability,then this issue may deserve more consideration moving forward becausemost glycerol produced today is a by-product of biodiesel and soapproduction. An additional consideration is the water grade used in theformulation. The indicated water grade was used to formulate most of theraw materials as concentrated solutions before assembling the reactions.As such, the water is confounding variable as the “complementing” watershown in Conditions #30 and #31 comprises only about 30% v/v of thefinal reaction volume. The better performing Red team of raw materialswas formulated with the USP grade water.

The role of raw material variability was also trended for precision. Theindividual coefficient of variations (% CV) for the twelve timepointswere compared for each of the thirty-six conditions (n=2replicates/condition/time point). There was a difference of almost 1.4%in the mean coefficient of variation between the best (mean, 1.15%;Condition 22) and worst (mean, 2.54%; Condition 32) of the thirty-sixconditions. The standard deviation of the mean % CV also similarlytracked by condition, with the spread between the best (STDEV, 0.55%;Condition 22) and worst (STDEV, 2.48%%; Condition 32) expanding to morethan a 1.9% difference. The gauge analysis did not show any dramatictrends in the mean/STDEV of the twelve % CV measurements, although therewere some modest individual trends on the mean % CV of the twelvemeasurements by CHAPS and BSA lot number. Interestingly, when the CHAPSand BSA lots were compared using an interaction analysis performed onthe STDEV's of the twelve measurements, these differences expanded. Forexample, the combination of CHAPS lot 7*BSA lot B (Condition 22) wasslightly better than CHAPS lot 1*BSA lot B (Condition 16). In contrast,CHAPS lot 1*BSA lot E (Condition 19) was considerably better than CHAPSlot 7*BSA lot E (Condition 25). One interesting aspect is that the BSAlot E (Diagnostic grade, lot 454) showed significant interaction effectsin a CHAPS-lot specific manner for both stability and precision metricin this study. By both metrics, this BSA lot E was considerably betterwith CHAPS lot #1 than when tested in combination with CHAPS lot #7.These results suggest has the combination of CHAPS lot and BSA lot usedin combination may a synergistic effect on calibrator function.

Regarding example 3, the Response Surface Design Stability Study, a typeof DOE, known as a response surface design, is useful for generating amap of a response to continuous factors and pinpointing a minimum ormaximum response within some specified design space. A popular type ofresponse surface design is the central composite design which combines afactorial design with center points and axial points. The axial pointsare located a specific distance outside the factor range explored in thefactorial design. For a three factor design, it may helpful to conceiveof the axial points residing on a sphere that fully engulfs a cube thatshare a common center point in three dimensions (see FIG. 22A-22B).Thus, the central composite design allows the experimenter to explorefive levels of each factor, namely low axial, low factorial, centerpoint, high factorial, and high axial. Here, a rotatable centralcomposite design was utilized to explore five raw materialconcentrations for four different raw materials (technically, it wasjust three actual raw materials, but the Tris buffer was independentlyvaried in two dimensions for both Tris buffer concentration and protonconcentration [pH]). The full factorial portion of the experimentallowed the design space around the standard concentrations to beexplored. The axial point portion of the experiment allowed extreme highand low concentrations to be surveyed, including the potential to“test-to-failure” using low (non-zero) concentrations for two rawmaterials, CHAPS and DTT. JMP modeling using the “response surfacedesign” fit modeling functionality was utilized to analyze the data.

The CHAPS concentrations were surveyed in a linear concentration rangewith a standard concentration of 4.76 mM used as the center pointconcentration. The low factorial concentration selected was 2.83 mM, areprise of a CHAPS concentration that demonstrated a deleterious effecton stability in a thirty-day Detergent Comparison study. The remainingCHAPS concentrations were chosen mathematically based on these twopre-designated concentrations. The DTT concentrations were surveyed in alinear concentration range with the standard concentration of 0.90 mMused as the high factorial concentration. The remaining four DTTconcentrations were selected to explore as broad a range ofconcentrations between axial concentrations (25-fold) as possible andengineering a potential “test-to-failure” concentration of 0.05 mM. Thecenter point concentration for the DTT was 0.65 mM. Similarly, the Trisbuffer concentrations surveyed used the standard concentration as thelow factorial point, and the pH range surveyed had the lower/upperspecifications of 7.95 and 8.05 incorporated as the midpoint and highfactorial concentrations, respectively.

The two raw materials which had the largest effect on stability were theCHAPS and DTT, but they showed different patterns of their respectivedeclines in stability. The axial low concentration of CHAPS demonstrateda sharp and steady decrease in stability observed over the course of thesix-month study starting at the first time point on Day 3, with acumulative 35% drop (e.g., see Condition 12 in FIG. 25). Consistent withthe earlier results from the Detergent Comparison study, there was alsoan immediate 10% drop-off in stability when CHAPS is surveyed at the lowfactorial concentration of 2.83 mM when assayed at conditions closelyapproximating the standard formulation (e.g., see Condition 4 in FIG.25). In contrast, the parallel condition using the high axial CHAPSconcentration of 6.69 mM are within the +/−3% specification (seeCondition #5 in FIG. 25). These results are consistent with a role forCHAPS micelle formation in maintaining optimal Lp-PLA2 stability. Theaxial low concentration of DTT showed an initial drop in stability ofabout 10% over the two initial two timepoints, spanning the first tendays of the study, before stabilizing (e.g., see Condition 11 in FIG.25). In contrast, the parallel conditions at the midpoint DTT condition(see Conditions 13/14 in FIG. 25) and at the high axial concentration ofDTT (see Condition 16 in FIG. 25) are both within the +/−3%specification. This immediate early effect for DTT might be interpreted,contextually, as a labile molecule with a short-half life (temperature-and pH-sensitive) having a role in the reduction some target moleculebefore rapidly undergoing decomposition in this slightly alkaline bufferconditions. For example, one candidate target molecule might be theCysteine-34 residue on the solvent interface of the bovine serumalbumin, a residue with a free sulfliydryl group prone to oxidation andmixed disulfide formation. Based on JMP modeling of this date set, theJMP prediction profiler predicted optimal concentrations for thefollowing raw materials: The profile predicted optimal stability, (1),trending toward 6.69 mM CHAPS (p=0.004), (2), trending toward a maximaat 0.70 mM DTT (p value <0.0001 for DTT*DTT) and, (3), trending towardpH 8.05 (pH as an effector was only statistically significant whenmodeled as part of an quadratic interaction with CHAPS or Time; see FIG.28). Surprisingly, the buffer's concentration, which spanned aconcentration range of 5-85 mM, was not a statistically significanteffector of stability in this study.

Several anecdotal trends with respect to precision were observed in theResponse Surface Design stability study as a function of raw materialconcentrations, in spite of the fact that the JMP modeling did not showthem to be statistically significant effectors. The highest bufferconcentration (85 mM) surveyed may improve calibrator precision. This isintriguing because buffer concentration appeared to have no effect onstability, per se, but it may have an effect on precision. Additionally,the axial high concentration tested for CHAPS (8.62 mM) may actuallyworsen calibrator stability slightly. Considering the axial low protonconcentration (pH 8.18) for potential precision improvements may also bean interesting future experimental direction to explore (also, see thepH effects on precision in below).

Regarding Example 4, the Buffer/BSA Survey Stability Study, thisBuffer/BSA survey stability study merged two distinct experimentalgoals. The first goal was to test various permutations of the Trisbuffer used in the calibrator formulation to parse out the importantaspects of the both the raw material composition and the process forobtaining optimal stability and precision. The permutations includeddifferent combinations of different buffer starting raw materials,different buffer concentrations, and process changes involving pre-pHprocess step, starting pH, and the final pH. A formulation condition wasincluded in this study which mimics previous formulation materials andprocedural process. A second goal was to further study the effects oncalibrator stability and precision contributed by different grades/lotsof Probumin BSA, including Universal Grade, Diagnostic Grade and FattyAcid-Free Grade. The same lots of both the Universal Grade BSA andDiagnostic Grade BSA that were previously tested in the context ofdifferent vendors' CHAPS raw materials in the Material Variationstability study are surveyed here in the context of a fourth distinctlot of Dojindo CHAPS detergent (L/N DC862). For comparison purposes,each of the calibrator formulations were stored both frozen (−70°degrees Celsius) and at refrigerated temperatures (4-8° degrees Celsius)and analyzed in parallel at each time point over the course of fourmonths.

In general, the percent stability was very good across all the buffercomposition and process permutations tested. The gauge trendingindicates that the buffer composition did not make much of differencewith regard to stability. With regard to buffer composition, the Trisbase and the Tris-Hydrochloride give essentially equivalent stability.The Tris base:hydrochloride and the pre-formulated Tris base solutionboth seem to give a slight amount of over-recovery. Very smalldifferences were observed as starting pH and final pH as most of thetrend lines fall well within the specification. At both temperatures,the biggest effectors of stability appear to be day-to-day variation andBSA lot number. In addition, this study seems to indicate an increasingpercent stability as a function of increasing buffer concentration, atleast with the single lot of Teknova-formulated buffer surveyed here. Incontrast, no effect was observed on percent stability in the ResponseSurface Design study as the buffer concentration was increased.

The Buffer/BSA stability study was also analyzed for effects onprecision. At both temperatures, the best precision was obtained at pH8.15 (both starting pH and final pH) relative to the other pH's 8.00 and7.90. It should be noted that the Tris buffers prepared at pH 8.20 areconfounded by their participation in the BSA survey portion of theexperimental design as well as the fact that the final pH (8.00)different from the pre-pH (8.20). While the low axial pH sample (8.18)in the Response Surface Experiment showed admirable precisionperformance in that study, it was not definitively established as thebest formulation condition for precision. In addition, it was hamperedby the fact that only one formulation was tested in the Response SurfaceDesign study at pH 8.18. The Response Surface Design, on the other hand,had three formulation conditions with a pH set at 8.15. At least in thecontext of this study, calibrator pH was also shown to have an effect oncalibrator precision. In addition, the trending by gauge analysisindicated that BSA lot 454 had the best precision with this lot of CHAPS(L/N DC862) in this study at both temperatures. In the MaterialVariation stability study, lot 454 (lot E in that study) showed goodprecision with Sigma CHAPS lot (L/N 018K53003) used in that study, butit showed inferior precision with the Dojindo CHAPS lot (L/N CY785)used. Conversely, the best BSA lot number in that study (referred to aslot 13 in the Material Variation study) showed only average precisionperformance in this study (referred to as lot 693 in this Buffer/BSAsurvey study). Taken together, these results suggest that BSA lot numbermay play a role in calibrator precision, and there might be synergisticeffects between the BSA lot and the CHAPS lot used in a given build.

The four real-time stability examples described above summarize theeffects of raw material variability and raw material solutionconcentration surveyed in the calibrator matrix for an ELISA PLAC kit.Based on the results of these studies, the following may improve therobustness of the calibrators with respect to stability and precisionand may provide a path for extending an expiration date of a kit. Giventhat calibrators may be used in any kit (e.g. in an Auto-CAM test), anyfuture improvement of calibrator performance may be tested in a varietyof assays, including mass and enzymatic assays, to ensure compatibility,or, alternatively, to fully assess the specific needs of the individualassays with respect to calibrator raw material quality/concentration.The following recommendations include suggestions for incoming qualitycontrol analytical testing, in-process test methods and manufacturingvalidations to maintain, extend and improve calibrator shelf life forthe family of Lp-PLA2 analyte-based products.

The CHAPS detergent's quality and its effective solution concentrationmay be an important quality parameter, and the CHAPS detergent may be animportant raw material in a calibrator formulation. Collectively,micelle formation may be important (or essential) for maintainingLp-PLA2 stability. As discussed above, a trace contamination of CHAPSpreparations with their precursor molecules may have deleterious effectson micelle formation of CHAPS. All the lots of CHAPS tested from a givenvendor are clearly not functionally equivalent in stability performancein our assay, and it is likely that only certain purities of CHAPS maymeet our quality requirements even if they meet the all the vendor'sspecifications. One possibility is to develop an analytical assay as anincoming quality control test method. This analytical method would bethe most facile approach assuming that performance can be shown todepend on control of one or more typical analytical specifications. Suchanalytical specifications may be one or more of, (1), sufficient to bepredictive of CHAPS performance in the calibrator matrix and, (2), begenerally applicable, if the detergent should need to be sourced from asecondary vendor. A second possibility is to establish a test method forqualifying performance of CHAPS for use in the calibrator itself similarto the test method used for qualifying BSA. This approach might be moretime-consuming, but, nevertheless, it has appeal because it will, (1),provide direct evidence given that the detergent is qualified in themanner in which it will ultimately be used, (2), test the detergent inthe context of the other calibrator components, including the BSA, and,(3) allow side-by-side comparison of performance to backlot(s) of CHAPSthat will be run as contemporaneous controls. In addition, there may bedistinct advantages to using the same lot of CHAPS in antigenpurification and the conjugate formulation.

The CHAPS concentration-dependent effects on stability might be a goodopportunity for the application of robust design principles (see FIG.45A). The CHAPS shows a sharp drop-off in stability performance atconcentrations less than 2.83 mM. The characterization of the CHAPSconcentration-dependent effects indicate a plateau in the stabilityresponse above the standard concentration of 4.76 mM with the caveatthat the precision got worse at the highest concentration tested of 8.62mM. The best overall results seem to be achieved at the intermediateconcentration of 6.69 mM where the stability and the precision are bothexcellent. Response of stability is predicted to change very little withsmall changes in CHAPS concentration around 6.69 mM at the plateau andthe variability is minimized due to the excellent % CV. (see FIG. 45B).

The Bovine Serum Albumin may be an important raw material in someformulations. The lot of BSA used can have an effect on both stabilityand precision of the calibrator. Similar to the CHAPS detergent, the BSAseems to have performance variability that is seemingly more dependenton the lot of raw material chosen than the grade of the material from agiven vendor. Some anecdotal and indirect evidence suggests that theremay be larger differences in performance variability of the BSA productbetween vendors. In addition, the evidence presented here suggests thatthe BSA and the CHAPS may work synergistically together to affectcalibrator performance. In addition to the typical BSA productspecifications provided (e.g., percent purity, percent protein, pH,sodium and chloride content, IgG contamination, endotoxin level andproteolytic activity), there are a number of well-known heterogeneitiesin BSA preparations that may affect performance. These might includealbumin/SH ratio, N-F transition, secondary and tertiary structures,degree of polymerization, polymer profile, heterologous and homologouspolymers, immunochemical protein contaminant profile, pl, fatty acid andlipid profiles and hormone profile. Given the complexity of the BSApurity profile and the unclear relationship in how all theseheterogeneities correlate with calibrator performance (if at all), therecommendation here is to maintain the current test method of qualifyingthe BSA for use in the calibrators, at the very minimum. One lot of BSAwas utilized in three of the stability studies presented here, and thislot of BSA showed consistently good performance relative to the some ofthe other tested lots of BSA. The results presented here suggest thatthe test method for BSA may be best assayed in the context of the otherraw materials to be used in future builds.

DTT is typically used in formulations to maintain the disulfide bonds inprotein in a sufficiently reduced state so as to facilitate correctfolding while simultaneously avoiding protein aggregate formation.Unfortunately, the DTT is a labile reagent that sensitive to bothtemperature and pH. In the studies reported here, the DTT concentrationseemingly has a threshold effect. The stability response shows adrop-off in stability at the lowest DTT concentration tested in theResponse Surface Design study. The results presented here suggest thatfunctional reducing agent is required for calibrator functionality, atleast in the early stages. When DTT from two separate vendors wassurveyed in the Material Variation study, they functioned comparably. Inthis study, the functional sulthydryl in calibrator preparations werequantified using the well-known assay using the Ellman's reagent withfreshly-prepared free cysteine as the standard. Fresh DTT reagent fromboth Sigma and BioVectra showed comparable suithydryl activity whenassayed on Day 1 of the study (42.5% and 48.4%, respectively, of theinitial targeted concentration of 0.95 mM). Although not observed herein this study, there have been anecdotal reports of DTT from certainmanufacturers not having the same specific activity from lot-to-lot atthe time of assay. The test grade of DTT screened in the MaterialVariability study was a special cGMP grade available from BioVectra.There may be an opportunity to improve lot-to-lot performance byvalidating the use of the cGMP grade DTT in the calibrator diluentformulation. In light of some of the previously reported process issueswith dissolving the DTT in the presence of high concentrations of BSA,there may be some opportunity to improve and standardize the formulationprocess by adding freshly-thawed DTT from a concentrated frozensolution. An in-process test method to evaluate the functionalsulthydryl activity on the stock DTT reagent and/or on the WIPcalibrator diluent could be implemented using the Ellman's reagent priorto the addition of antigen. At first glance, the Ellman's reagent seemsto be functional in the context of the calibrator diluent formulation(see Section 8.3.1). The adoption of an in-process test method couldinvolve setting up a capability study to establish a lower specificationlimit for DTT specific activity at some fixed time after completion ofthe calibrator diluent formulation work.

Raw materials may be tested prior to use. The test grade glycerolsurveyed in this study showed a strong effect in giving “over-recovery”of calibrator stability relative to the standard grade. While this doesnot suggest that the standard grade of glycerol is the root cause of thecalibrator instability problem, there may be an opportunity to utilize ahigher quality glycerol reagent to eliminate any possibility of thismanifesting itself as an issue due to lot-to-lot variability. Given thatalmost all glycerol is produced as a byproduct of other manufacturingprocesses, this type of glycerol can contain animal fats such as beeftallow, and vegetable oils such as coconut, palm kernel, cottonseed, andsoybean, which may lead to inferior product stability and significantimpurities. One vendor (Dow Optim™ synthetic glycerine) manufactures“synthetic” glycerol that is of USP/cGMP grade and is designed for usein pharmaceutical and biotechnology applications. This pharmaceuticalgrade synthetic glycerol may be used in the calibrator diluentformulation. Similarly, use of USP/cGMP grade water and cGMP gradehydrochloric acid may be useful. There may also be formulation processchanges related to the glycerol that may lead to improvements incalibrator stability, such as adding the glycerol prior to filtration.

An effect of pH on precision was observed in the Buffer/BSA Survey studyis also notable. There was a noticeable trend of the precision improvingfrom pH as it became slightly more alkaline, adjusting from pH 7.90 topH 8.00 to pH 8.15. It is not clear if the starting pH being identicalto the final pH is an important parameter: When comparing individualformulations, the two best conditions for precision were Condition 17(starting pH8.15=final pH8.15) and Condition 18 (starting pH8.20 finalpH8.00). Given that the same trends were observed both at both thefrozen and refrigerated storage temperature, it is unclear what theexact basis for this precision improvement might be. It is conceivablethat precision improvement is assay condition-based and not necessarilythe result of improved Lp-PLA2 stability, but these two possibilitiesare not mutually exclusive. Irrespective of the mechanism, there may bean opportunity to improve the precision of a Lp-PLA2 calibrator functionby adjusting the calibrator pH to 8.15.

The results of the four studies summarized in this report have increasedthe understanding of which raw materials may be important to theachievement of good stability performance and they suggest ranges andoptimal values to maximize stability and minimize variation. Suchchanges may extend calibrator shelf life and result in an overallimprovement in product performance for assays, such as a PLAC ELISA andAuto-CAM assay. These improvements may require one or more of thefollowing: refining a calibrator formulation, developing an incomingquality control analytical testing and/or in-process test methods,performing an additional manufacturing validation using high quality(cGMP) raw materials, implementing an additional process control and/orperforming capability studies to define specifications. Such aquality-by-design approach may provide tangible product stabilityimprovements so that product shelf life can eventually be extended tonine months, ten months, eleven months, twelve months or more thantwelve months expiration dating.

In general, a recombinant Lp-PLA2 may have between about 70 and 100%identity with the amino acid sequence of human Lp-PLA2. For reference,listed below are amino acid sequences of one variation of human Lp-PLA2.

For example, recombinant Lp-PLA2 may be recombinant humanPlatelet-Activating Factor Acetylhydrolase/PAFAH, and may be producedwith a mammalian expression system (e.g., in human cells). The targetprotein may be expressed with sequence (Phe22-Asn441) of Human PAFAHfused with a polyhistidine tag at the C-terminus (e.g., VDHHHHHH (SEQ IDNO: 4)). In general, Lp-PLA2 may be referred to as Platelet-ActivatingFactor Acetylhydrolase, PAF Acetylhydrolase,1-Alkyl-2-Acetylglycerophosphocholine Esterase,2-Acetyl-1-Alkylglycerophosphocholine Esterase, Group-VIIA PhospholipaseA2, gVIIA-PLA2, LDL-Associated Phospholipase A2, LDL-PLA(2), and PAF2-Ac. SEQ ID NO: 1, below, provides one example of recombinant Lp-PLA2that may be used as described herein:

rLp-PLA2.1, SEQ ID NO: 1:FDWQYINPVAHMKSSAWVNKIQVLMAAASFGQTKIPRGNGPYSVGCTDLMFDHTNKGTFLRLYYPSQDNDRLDTLWIPNKEYFWGLSKFLGTHWLMGNILRLLFGSMTTPANWNSPLRPGEKYPLVVFSHGLGAFRTLYSAIGIDLASHGFIVAAVEHRDRSASATYYFKDQSAAEIGDKSWLYLRTLKQEEETHIRNEQVRQRAKECSQALSLILDIDHGKPVKNALDLKEDMEQLKDSIDREKIAVIGHSFGGATVIQTLSEDQRFRCGIALDAWMFPLGDEVYSRIPQPLFFINSEYFQYPANIIKMKKCYSPDKERKMITIRGSVHQNFADFTFATGKIIGHMLKLKGDIDSNVAIDLSNKASLAFLQKHLGLHKDFDQWDCLIEGDDENLIPGTN INTTNQHIMLQNSSGIEKYN

Another variation of a human recombinant Lp-PLA2 having an N-terminalHis-tag fused to the sequence is shown in SEQ ID NO: 2, below.

rLp-PLA2.2, SEQ ID NO: 2:  MGHHHHHHSGSEFELRRQ-FDWQYINPVAHMKSSAWVNKIQVLMAAASFGQTKIPRGNGPYSVGCTDLMFDHTNKGTFLRLYYPSQDNDRLDTLWIPNKEYFWGLSKFLGTHWLMGNILRLLFGSMTTPANWNSPLRPGEKYPLVVFSHGLGAFRTLYSAIGIDLASHGFIVAAVEHRDRSASATYYFKDQSAAEIGDKSWLYLRTLKQEEETHIRNEQVRQRAKECSQALSLILDIDHGKPVKNALDLKFDMEQLKDSIDREKIAVIGHSFGGATVIQTLSEDQRFRCGIALDAWMFPLGDEVYSRIPQPLFFINSEYFQYPANIIKMKKCYSPDKERKMITIRGSVHQNFADFTFATGKIIGHMLKLKGDIDSNVAIDLSNKASLAFLQKHLGLHKDFDQWDCLIEGDDENLIPGTN INTTNQHIMLQNSSGIEKYN

Another variation of Recombinant Lp-PLA2 (Phe22˜Asn440) may be expressedin E. coli. This variation is a mouse-derived recombinant protein. Thus,in general, and of the recombinant Lp-PLA2 proteins described herein maybe non-human derived Lp-PLA2 proteins (e.g., mouse, rat, dog, horse,etc.). For example, the target protein may be fused with N-terminalHis-Tag. The sequence is listed in SEQ ID NO: 3.

rLp-PLA2.3, SEQ ID NO: 2:  MGHHHHHHSGSEFELRRQ-FHWQDTSSFDFRPSVMFHKLQSVMSAAGSGHSKIPKGNGSYPVGCTDLMFGYGNESVFVRLYYPAQDQGRLDTVWIPNKEYFLGLSIFLGTPSIVGNILHLLYGSLTTPASWNSPLRTGEKYPLIVFSHGLGAFRTIYSAIGIGLASNGFIVATVEHRDRSASATYFFEDQVAAKVENRSWLYLRKVKQEESESVRKEQVQQRAIECSRALSAILDIEHGDPKENVLGSAFDMKQLKDAIDETKIALMGHSFGGATVLQALSEDQRFRCGVALDPWMYPVNEELYSRTLQPLLFINSAKFQTPKDIAKMKKFYQPDKERKMITIKGSVHQNFDDFIFVTGKIIGNKLTLKGEIDSRVAIDLTNKASMAFLQKHLGLQKDFDQWDPLVEGDDENLIPGSPF DAVTQVPAQQHSPGSQTQN

Any of the recombinant Lp-PLA2 proteins described herein may include oneor more polymorphisms, and in particular known polymorphisms for humanLp-PLA2.

Any of the solutions described herein, and particularly the solutionsincluding recombinant Lp-PLA2 having a long shelf life may be used as astandard, control, calibrator or re-calibrator. For example, any ofthese solutions may be used to calibrate an assays, such as an assay fordetection of Lp-PLA2 activity and/or amount, or it may be used as acontrol (e.g., a positive control) for an assay for detection of Lp-PLA2activity or amount.

Positive controls are often used to assess test validity. For example,to assess a test's ability to detect a disease (its sensitivity), thenit can be compared against a different test that is already known towork. The well-established test is the positive control, since it hasalready been established to work. For example, in an enzyme assay tomeasure the amount of an enzyme (e.g., Lp-PLA2) in a set of extracts, apositive control may be an assay containing a known quantity of thepurified enzyme (e.g., recombinant Lp-PLA2) while a negative controlwould contain no enzyme (e.g., a predetermined concentration ofrecombinant Lp-PLA2 of zero). The positive control should give a largeamount of enzyme activity, while the negative control should give verylow to no activity. If the positive control does not produce theexpected result, there may be something wrong with the, experimentalprocedure, and the experiment may be repeated. For difficult orcomplicated experiments, the result from the positive control can alsohelp in comparison to previous experimental results. For example, if thewell-established disease test was determined to have the sameeffectiveness as found by previous experimenters, this indicates thatthe experiment is being performed in the same way that the previousexperimenters did. Multiple positive controls may be used, which mayalso allow finer comparisons of the results (calibration, orstandardization) if the expected results from the positive controls havedifferent sizes. For example, in the enzyme assay discussed above, astandard curve may be produced by making many different samples withdifferent quantities of the enzyme.

In general, terms such as calibration, calibrators, standards,standardization, reference, control, and re-calibrator are usedconsistent with the meanings as described in the InternationalVocabulary of Metrology-Basic and General Concepts and Associated Terms(VIM) (JCGM 200:2012), which is herein incorporated by reference in itsentirety.

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.For example, as used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms“comprises” and/or “comprising,” when used in this specification,specify the presence of stated features, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, steps, operations, elements, components, and/orgroups thereof. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items and may beabbreviated as “/”.

Although the terms “first” and “second” may be used herein to describevarious features/elements, these features/elements should not be limitedby these terms, unless the context indicates otherwise. These terms maybe used to distinguish one feature/element from another feature/element.Thus, a first feature/element discussed below could be termed a secondfeature/element, and similarly, a second feature/element discussed belowcould be termed a first feature/element without departing from theteachings of the present invention.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about” or “approximately,” even if theterm does not expressly appear. The phrase “about” or “approximately”may be used when describing magnitude and/or position to indicate thatthe value and/or position described is within a reasonable expectedrange of values and/or positions. For example, a numeric value may havea value that is +/−0.1% of the stated value (or range of values), +/−1%of the stated value (or range of values), +/−2% of the stated value (orrange of values), +/−5% of the stated value (or range of values), +/−10%of the stated value (or range of values), etc. Any numerical rangerecited herein is intended to include all sub-ranges subsumed therein.

Although various illustrative embodiments are described above, any of anumber of changes may be made to various embodiments without departingfrom the scope of the invention as described by the claims. For example,the order in which various described method steps are performed mayoften be changed in alternative embodiments, and in other alternativeembodiments one or more method steps may be skipped altogether. Optionalfeatures of various device and system embodiments may be included insome embodiments and not in others. Therefore, the foregoing descriptionis provided primarily for exemplary purposes and should not beinterpreted to limit the scope of the invention as it is set forth inthe claims.

The examples and illustrations included herein show, by way ofillustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of various embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

What is claimed is:
 1. A value-assigned solution oflipoprotein-associated phospholipase A2 (Lp-PLA2) for use with anLp-PLA2 assay as a control, standard, calibrator or re-calibrator, thevalue-assigned solution having a shelf-life of greater than 4 months,the value-assigned solution comprising: a first predeterminedconcentration of a recombinant Lp-PLA2 in a low-salt buffer solutionhaving a salt concentration below about 1 M, wherein the low-salt buffersolution comprises a detergent forming a plurality of micelles thatstabilize the recombinant Lp-PLA2.
 2. The value-assigned solution ofclaim 1, further comprising a second detergent to prevent aggregation ofthe recombinant Lp-PLA2.
 3. The value-assigned solution of claim 2,wherein the second detergent comprises a non-ionic detergent.
 4. Thevalue-assigned solution of claim 2, wherein the second detergentcomprises a polysorbate detergent.
 5. The value-assigned solution ofclaim 1, wherein the detergent is above a critical micelle concentration(CMC).
 6. The value-assigned solution of claim 1, wherein the detergentcomprises a cholate detergent.
 7. The value-assigned solution of claim1, wherein the detergent comprises CHAPS.
 8. The value-assigned solutionof claim 1, wherein the low-salt buffer solution comprises anon-chaotropic salt.
 9. The value-assigned solution of claim 1, whereinthe low-salt buffer solution comprises one or more of: NaCl and anacetate salt.
 10. The value-assigned solution of claim 1, wherein thelow-salt buffer solution includes a protein buffered matrix.
 11. Thevalue-assigned solution of claim 1, wherein the low-salt buffer solutionincludes a pH buffer.
 12. The value-assigned solution of claim 1,wherein the low-salt buffer solution includes Tris as a pH buffer.
 13. Avalue-assigned solution of lipoprotein-associated phospholipase A2(Lp-PLA2) for use with an Lp-PLA2 assay as a control, standard,calibrator or re-calibrator, the value-assigned solution having ashelf-life of greater than 4 months, the value-assigned solutioncomprising: a first predetermined concentration of a recombinant Lp-PLA2in a low-salt buffer solution having a salt concentration below about 1M, wherein the low-salt buffer solution comprises a detergent forming aplurality of micelles that stabilize the recombinant Lp-PLA2 and asecond detergent to prevent aggregation of the recombinant Lp-PLA2. 14.A lipoprotein-associated phospholipase A2 (Lp-PLA2) kit for use with anLp-PLA2 assay, the kit having a shelf-life of greater than 4 months, thekit comprising: a first value-assigned solution comprising a firstpredetermined concentration of a recombinant Lp-PLA2 in a first low-saltbuffer solution having a salt concentration below about 1 M, wherein thefirst buffer solution comprises a cholate detergent forming a pluralityof micelles that stabilizes the recombinant Lp-PLA2, a protein bufferedmatrix, a pH buffer and a preservative; and a second value-assignedsolution comprising a second low-salt buffer solution having a saltconcentration below about 1 M, wherein the second buffer solutioncomprises a plurality of micelles of the cholate detergent.
 15. The kitof claim 14, wherein the cholate detergent comprises CHAPS.
 16. The kitof claim 14, wherein the preservative comprises sodium azide.
 17. Thekit of claim 14, wherein the protein buffered matrix comprises bovineserum albumin (BSA).
 18. The kit of claim 14, wherein the first buffersolution comprises a second detergent to prevent aggregation of therecombinant Lp-PLA2.
 19. The kit of claim 14, wherein the second buffersolution comprises a second detergent comprising a polysorbatedetergent.
 20. A lipoprotein-associated phospholipase A2 (Lp-PLA2) assayhaving recombinant value-assigned solutions having a shelf-life of morethan 4 months, the assay comprising: a plurality of value-assignedsolutions each comprising a predetermined concentration of a recombinantLp-PLA2 in a low-salt buffer solution having a salt concentration belowabout 1 M, wherein the buffer solution comprises a cholate detergentforming a plurality of micelles that stabilizes the recombinant Lp-PLA2;a solution comprising an agent that interacts with Lp-PLA2 to produce adetectable signal; and a wash buffer.
 21. The assay of claim 20, furthercomprising a solid phase support configured to bind Lp-PLA2.
 22. Theassay of claim 20, wherein the agent comprises a report antibodyspecific to Lp-PLA2.
 23. The assay of claim 20, wherein the low-saltbuffer further comprises a second detergent to prevent aggregation ofthe recombinant Lp-PLA2.
 24. The assay of claim 20, wherein the cholatedetergent is above a critical micelle concentration (CMC) for thecholate detergent.
 25. The assay of claim 20, wherein the cholatedetergent comprises CHAPS.
 26. The assay of claim 20, wherein thelow-salt buffer solution comprises a non-chaotropic salt.
 27. The assayof claim 20, wherein the low-salt buffer solution includes a proteinbuffered matrix.
 28. The assay of claim 20, wherein the low-salt buffersolution includes bovine serum albumin (BSA) as a protein bufferedmatrix.
 29. The assay of claim 20, wherein the low-salt buffer solutionincludes a pH buffer.